Men have poor access to sexual and reproductive health (SRH) services globally, particularly in low- and middle-income countries (LMICs). Nevertheless, in LMIC and high-income countries (HICs), low SRH utilisation happens on account of several factors, such as individual, health system-related, and sociocultural factors. Identifying and addressing men’s SRH service underutilisation remains essential to improving their sexual health and averting higher mortality and early morbidity associated with poor health seeking behaviour (HSB) among men.This narrative review identifies factors influencing whether men do or do not utilise SRH services in LMICs.We report on articles published in LMICs: Africa, Asia and South America.In this narrative review, we searched for quantitative and qualitative articles published between 2004 and 2021 from international databases, including Google Scholar, ScienceDirect, EBSCOhost, Scopus, PubMed, Medline, and reference lists of retrieved published articles.A total of 2219 articles were retrieved, from which 36 met the inclusion criteria. Factors contributing to poor uptake of SRH services by men included: a lack of access and availability of SRH services, poor health-seeking behaviour among men, and SRH facilities not being perceived as ‘male-friendly spaces’. Furthermore, our review reveals that decreased use of SRH services is attributed to factors such as a lack of focus on men’s SRH.The current underutilised state of SRH services calls for urgent implementation of evidence-based interventions. Identifying men’s SRH service inhibitors and enablers will assist programme managers and policymakers in designing SRH services tailored to their sexual health needs.Despite numerous global interventions to motivate men, the findings provide insight into the underutilisation of SRH services. The study also reveals the inadequate comprehensive investigation of men’s SRH service utilisation, especially older men, to comprehend men’s problems fully. Further research needs to be conducted on SRH issues, including vasectomy, mental health, and chronic conditions related to sexual and reproductive health. The analysis can assist SRH policymakers and program managers in strengthening the policies to motivate men to engage better with SRH services. These factors include the lack of focus on men’s SRH by international programmes such as Sustainable Development Goals (SDGs) and Family Planning 2020 (FP2020), which largely focus on women and youth.16 Furthermore, SRH service provision is often fragmented or poorly structured for men’s health needs and is mostly needlessly expensive.16Despite men’s need for SRH services, most studies report underutilisation of SRH by men.The purpose of the review is to synthesise available evidence on factors influencing whether men do or do not utilise SRH services in low- and middle-income countries (LMICs). The review focused on LMICs because of their resource constraints which lead to poorer access and a higher unmet need for SRH services. This review aimed to answer our research question: ‘What factors influence whether men do or do not utilise SRH services in LMICs?’The non-systematic narrative literature review method was followed to identify relevant literature. The search included published peer-reviewed articles, reports, and grey literature. Peer-reviewed articles were searched in the following search engines and databases: Google Scholar, ScienceDirect, Scopus, and EBSCOhost. Additionally, electronic databases: CINHAL, PubMed, Medline, Academic Search Complete, Health Source – Consumer Edition, Health Source: Nursing/Academic Edition, and MEDLINE, electronic journals, and reference lists of retrieved published articles were also searched.The following Medical Subject Headings (MeSH) terms were used to retrieve relevant articles for this review: ‘sexual and reproductive health services,’ ‘men,’ ‘factors influencing or inhibiting/facilitating SRH service utilisation,’ and the ‘utilisation of SRH services by men in LMICs’. We also used keywords from the extracted articles to help narrow the focus. To cover SRH services comprehensively, studies were also searched based on individual SRH services, for example, using condoms, vasectomy services, HIV services, prostate cancer (PC), family planning (FP), MMC, and infertility. Studies that investigated Health Care Workers’ (HCW) knowledge and perceptions about men’s use of SRH services were also included.6 The database was constructed to extract important dimensions of factors influencing SRH service utilisation. We extracted only relevant results about populations eligible for inclusion. For example, where data were collected from males and females, we extracted only data presented for males. Hence, studies focused exclusively on women were excluded.This review included quantitative and qualitative papers published in English between 2004 and 2021. Studies conducted in 2004 were sourced because the World Health Organization (WHO) first recognised the immense global health burden associated with poor quality of care for people’s SRH in 2004.We included studies reporting on factors influencing SRH service utilisation by men aged 15 years and above. We aimed to target studies focusing on older men because the literature review indicated less focus on SRH service utilisation among this population. Therefore, studies reporting exclusively on the factors influencing adolescent SRH service utilisation were also excluded. However, studies including subjects aged 15 years and above were included because of overlapping reporting on the studies focusing on this review’s research question and the paucity of studies focusing exclusively on older men. For example, identified studies focused on ages 15 years and above, 15–25 years, or 15–69 years. The research focusing on SRH service utilisation among adolescents is over-researched, as approximately 90% of studies identified in this review focused exclusively on adolescents. Studies reporting on the interventions and the evaluation of interventions to improve SRH utilisation, clinical decision-making algorithms, studies conducted in high-income countries (HICs), and studies not reporting on barriers or facilitators of SRH utilisation were also not considered.The database search generated 2219 records, including grey literature, health services reports, original research articles, theses, and dissertations. Full-text articles were derived from different databases and analysed separately by two researchers. After that, the researchers agreed by collating all relevant articles for the study. On a review of titles, 2042 were excluded because of duplication, unrelated to this narrative review topic, or exclusively focused on adolescents and women. On a review of abstracts, 96 papers were excluded because they reported on the evaluation of SRH services, management of SRH problems, or male involvement in partner FP. These policies and documents did not report on factors facilitating or hindering SRH service utilisation. The latter were systematic reviews.Of 73 potentially relevant full-text papers, 37 were excluded because the results did not yield factors hindering or facilitating SRH service utilisation. Thirty-six papers met the inclusion criteria and were captured in the database. Secondary to the heterogeneity of papers, we present a narrative synthesis describing study characteristics and key findings. We further summarise overarching themes and the consistency of key findings. A database was constructed to summarise the studies identified for the review. The following information was captured in the database:  – authorApproval to conduct the study was provided by the Biomedical Research Ethics Committee (BREC) University of KwaZulu-Natal (number: BE 347/19).Of 2219 articles retrieved, 36 studies met the inclusion criteria for our narrative literature review. The review includes quantitative, qualitative, and mixed methods studies that reported evidence on factors influencing SRH service utilisation by men. 28 Four (11%) studies were conducted in Kenya32 three (8%) were conducted in Ghana,35 Nigeria,38 and Nepal41 and two (6%) were conducted in Zimbabwe43 and Uganda.45 One study was conducted in Rwanda,46 Swaziland,47 Papua New Guinea,48 Lesotho,49 Mexico,50 Philippines,51 Ethiopia,52 Egypt,53 Myanmar,54 and Lao.55 The included studies targeted men aged 15 years and above. Most studies (55%) exclusively targeted men, and 45% focused on both men and women. Eighty-six percent of studies targeted men aged 26 years and above. The remaining 14% targeted men aged between 15 and 25 years.Most studies were conducted in South Africa (22%).Most studies that met the inclusion criteria were conducted qualitatively and employed either focus group discussions (FGDs) or in-depth interviews (IDIs), and some both FGDs and IDIs (24%). Twenty-five percent of quantitative studies employed a cross-sectional design, and two employed the National Health and Demographic Survey (NHDS). The latter (16%) used mixed methods. Only one mixed study included randomised control trial (RCT) data. Forty-nine percent of studies were conducted in both urban and rural settings. In these settings, data were collected in towns and townships, rural villages where households were visited, and some participants were recruited on the streets. In studies conducted in public and private-sector health facilities , data were collected in private rooms or offices in community centres, non-governmental organisations (NGOs) sites, and district health offices. The SRH issues identified were vasectomy; FP, MMC; condom use; management and prevention of STIs; HIV services; PC screening, and erectile dysfunction (ED).57 The barriers to men’s SRH service utilisation can be categorised into health service system factors, lack of knowledge, individual and/or personal factors, sociocultural and religious, socioeconomic, and geographical factors, as detailed further.Several barriers have been associated with whether men do or do not utilise SRH services. In the same vein, some studies revealed that, poor health-seeking behaviour could be a barrier to SRH services utilisation.32; the awkwardness of the location of the SRH services52; long-waiting times because of lengthy queues,45 the fact is that SRH services globally, and especially in LMICs, are largely focused on the needs of women (of reproductive age) and offer little for boys and men.17 Further hindering health system factors include poor service quality and lack of materials such as condoms and medicines.45 Men may avoid using SRH services because of hostile and judgemental attitudes from female health service providers, especially towards young and unmarried men,32 and not having same-sex health workers. Some men felt embarrassed to discuss their health issues or be examined by female health workers.53 Notably, most studies that focused on the utilisation of SRH services such as contraceptives, STI and/or HIV services, and condoms and comprehensively on various services revealed a lack of privacy, respect, and potential breaches of confidentiality from the HCW at the health facilities as deterrent to men’s utilising of SRH services.55Amidst health service system factors contributing to underutilising SRH services, inconvenient or limited operating service hours43 In some studies, a lack of knowledge and understanding of HIV was evident when men often inferred their status from their female partners’ results.25 Lack of knowledge and reliable information about the benefits of MMC discouraged men as they claimed no benefits if they were already HIV-positive; had good hygiene; and were already practising other HIV-prevention methods such as the ‘Abstinence’ ‘Be faithful’ ‘Condomise’ (ABC) method.50 Furthermore, in previously non-circumcising communities in South Africa, men did not know that the MMC service was free at the local clinic or hospital.21 Secondary to a lack of knowledge, myths tend to impede the utilisation of SRH services. For example, post-circumcision, men opted not to use condoms after getting circumcised, citing reasons such as the circumcised penis would tear a condom; or putting a condom on an exposed circumcised penis would cause pain.41 In some studies, men associated circumcision with infertility.49 Equally, men often confuse vasectomy with castration and wrongly associate it with loss of libido, decreased sexual activity, and loss of masculinity.51Men’s underutilisation of SRH services was associated with a lack of knowledge and awareness of disease such as PC and HIV by SRH screening services.49 Post-circumcision myths, such as the inability to sexually satisfy partners and decreased penile sensitivity on a circumcised penis, also dissuaded certain men.30 Men were reluctant to utilise HIV services, fearing the possibility of testing positive, hence death related to HIV complications because of self-knowledge of infidelity. Men also feared the stigma associated with HIV, the possibility of being blamed and rejected by significant others such as partners, family members, and friends, and the pressure from employees to quit their job after diagnosis.54 Condom-use-associated barriers included poor quality of condoms, embarrassment when buying condoms, and perceived low risk.41 In Nigeria, some men were reluctant to screen for PC because there was no family history, and they did not believe they were at risk.38In this review, individual or personal factors are defined as behaviour and characteristics demonstrated by men which determined whether they utilised SRH services or not. Fear emerged as the dominant barrier among individuals as an obstacle to utilising SRH services. Among the studies focused on MMC, fear of post-operative complications such as pain, delayed recovery, infections, loss of morning erections, and the post-procedure abstinence period presented major barriers to MMC.47 Factors associated with culture, such as the threat to masculinity, deterred men from undergoing MMC. For example, non-circumcising communities presumed circumcision to be an alien culture or part of a foreign religion.30 Some men referred to undergoing MMC as tampering with God’s creation.50Most cultural and religious practises consider discussing sexual matters taboo. Therefore, the sensitivity of discussing SRH issues has hindered many men from accessing SRH services.35 In addition, the lack of access to condoms contributed to low utilisation.39 Taking time off work and losing income while, for example, waiting for the wound to heal post-MMC deterred some men from undergoing MMC.47 Furthermore, travelling costs to healthcare establishments also emerged as a significant deterrent as men lacked the money for transport.27 Health service utilisation is also compromised by high human geographic mobility as men constantly relocate from rural to urban areas in search of employment. Consequent to high mobility, negative interactions with healthcare providers, language barriers, and missed appointments may discourage health-seeking.58 This internal migration is historically coupled with the Apartheid system that restricted black South Africans from permanently settling in the urban areas where they were recruited to work in mines.59 Migration can hinder adherence to and continuity of healthcare for men who already had contact with health services.58Sexual and reproductive health service utilisation was hindered by their perceived high costs and related products such as vasectomy and condoms .60 Consequently, long distances and poor transport  to the health facility are barriers to accessing and utilising SRH services.52Health facilities are ordinarily concentrated in urban rather than rural areas; however, most regions in LMICs are rural.Factors facilitating men’s utilisation of SRH services are summarised into health service system, knowledge, individual and/or personal issues, and socioeconomic factors.53 Access to the right information about SRH services via advertisements such as pamphlets and radio or television programmes25 and support from healthcare providers also played a vital role in encouraging SRH service use by men.43Health workers’ welcoming and friendly attitudes and respect for men’s privacy and confidentiality motivated men to access and utilise SRH services.55 Men who knew that vasectomy was a low-risk procedure, with few complications and side effects and unlikely fail, were motivated to undergo the procedure to limit family size.51The benefits of undergoing MMC and using condoms, such as protection against diseases and improved hygiene, motivated many men to undergo the procedure.42 Furthermore, personal gain or prestige from research activities, such as free medical care, financial incentives, and a sense of responsibility from research, motivated men to utilise SRH services.42 Role modelling positive HIV status disclosure and adherence to antiretroviral therapy (ART) motivated men to engage in HIV treatment initiatives.43 Furthermore, an individual’s desire to limit family size encouraged some men to undergo a vasectomy as it was perceived as a permanent method with a low risk of complications, thus limiting the side effects of other female-controlled hormonal methods.51In some studies, men were motivated to perform MMC as they believed women were better satisfied sexually after circumcision. Wearing condoms was much easier after the foreskin had been removed.36Although vasectomy may be free in some LMICs, getting an appointment for the procedure can be a long process. However, men who can secure an appointment for the procedure or those who can afford to pay to counteract financial difficulties decided to undergo a vasectomy to limit family size and reduce expenses. Therefore, the dire socioeconomic state can be a motivating factor in undergoing vasectomy.50 However, further research is required to identify the reasons behind this. Furthermore, governments should make men’s SRH services affordable and accessible.This narrative review aimed to establish the factors influencing whether men do or do not utilise SRH services. In this review, only studies conducted in LMICs were included for synthesis. Identified studies were conducted mostly in the African regions  and only 10% were conducted in South Asia. Although studies were conducted in either rural or urban areas , findings in these settings remained comparable. Research indicates less focus on SRH issues such as PC screening, ED, and vasectomy services. The assumption may be less utilisation of these services because of high costs, as these SRH issues may be freely available only in some government health establishments in LMICs. Research in L and MIC is often conducted among unemployed and rural-based communities where men may not be able to afford such services, and sometimes they are against their culture and religious beliefs.45 Many men find it embarrassing to go to SRH services and consider it a very negative experience when they are seen, ridiculed, and disrespected by people known to them.55 These negative experiences fundamentally discourage SRH service utilisation by men.Fear plays a vital role in inhibiting men’s use of SRH services. The most common barriers related to personal factors included fear, embarrassment, and insecurity related to self-esteem and reputation. Men are discouraged from utilising SRH services such as MMC, vasectomy services, FP, STI and/or HIV, and PC screening.33 The threat to masculinity, especially secondary to misconceptions and myths, significantly deters men from utilising SRH services such as MMC or vasectomy. Men are often concerned about the inability to produce more children, the loss of manhood, and infertility.47 Despite the availability of services, poor utilisation of SRH remains associated with limited knowledge of the various available forms of SRH services. Lack of knowledge or awareness of SRH services such as vasectomies, screening for PC, STI and/or HIV testing, MMC, centres for counselling, and information provision contribute to men’s low utilisation of SRH services.45 Despite the many educational campaigns provided on various SRH services in the past, governments still need to roll out sustained ongoing campaigns at schools, workplaces, communities, and during weekends.The under-utilisation of SRH services is associated with a range of cultural barriers. For example, cultural and religious backgrounds perceive discussing sexual matters as taboo and deter men’s utilisation of SRH services.35 Such difficulties in accessing SRH services may be due to unemployment. In remote areas, several health facilities are far from the communities. Consequently, people in rural-based communities must walk long distances because of a lack of money and transport.60 Moreover, in some health establishments, healthcare workers’ conduct significantly influences whether men do or do not utilise SRH services. Men’s utilisation of SRH services is predominantly deterred by bad health workers’ attitudes and lack of privacy and confidentiality.45Certain studies reveal financial constraints as fundamental inhibitors in accessing and utilising SRH services such as vasectomies, PC services, and MMC. Although most SRH services are free in some LMICs, some men, especially from rural-based settings, allude to high cost as a hindrance to accessing SRH services.49 Knowledge is also instrumental in encouraging SRH service utilisation. Men who understand the benefits of MMC are motivated to perform the procedure.44 It is imperative also to note that men need the constant provision of SRH information, especially about the benefits and existence of various SRH services. In addition, professional and better treatment from healthcare workers encourages SRH service utilisation.25Amid studies that investigated enablers of SRH service utilisation, virility motivated men to undergo MMC. Post-MMC, some men claim to be stronger sexually and that wearing condoms is much easier.20 Low SRH utilisation contributes to high morbidity and mortality among men and indirect mortality among women. Low SRH service utilisation, such as MMC, indirectly contributes to high HIV infection rates. In South Africa, almost 20% of adults aged 15–49 years are estimated to be HIV positive, while 8.2 million people live with HIV.60 HIV infection rates and low uptake of MMC remain major concerns in LMICs.Despite the facilitating factors, most studies revealed that barriers to SRH service utilisation outweighed and require urgent attention. Some studies identified low utilisation of SRH services despite being available at low cost or no cost in some settings.48 Moreover, a positive trend in the uptake of HIV testing is well-documented. Meanwhile, a significant number of people still require testing.25 The effects of the HIV pandemic have created a shortage of human resources while plunging most countries into financial crises and knocking down national treatment implementation plans.24 High HIV infection rates in the general population indicate the need for various effective prevention measures, such as high uptake of circumcision, to reduce the incidence of HIV infections.22Nevertheless, multiple studies suggest that almost 80% coverage of MC would be necessary to impact HIV significantly and decrease incidence by at least 35%.19 High sexually transmitted infections among men may also be linked to STD screening programmes that unilaterally focus on women during the reproductive period. Men are less likely to seek or receive regular screening for SRH care, and most countries lack STD screening programmes to support regular screening in men.52 A multi-country HIV study conducted in sub-Saharan Africa found that men were less likely to take advantage of the programme and test for HIV compared to women. In some instances, where an equal proportion of men and women used HIV testing services, men would only get tested for HIV after becoming severely ill.43 The gender gap in health-seeking behaviour among men has been associated with the masculinity factor. Masculinity is a patriarchal, culturally promoted status quo held by men.23Furthermore, limited knowledge, low-risk perceptions, unknown HIV statuses, and the high rates of STDs in some LMICs illustrate less condom use.33 Despite excellent health policies to improve SRH service utilisation, many LMICs still face low SRH service uptake leading to the inability to exercise fertility preferences. Consequently, fertility remains high despite substantial FP policies that allow modern contraceptive use without consent from a partner or parent, especially in communities experiencing poor socioeconomic status.51 Therefore, identifying barriers to contraceptive uptake is warranted, especially in areas with high fertility. In LMICs such as Uganda, persistent high fertility contributes to high maternal morbidity and mortality, and a rapidly growing population.Secondary to cultural beliefs, political barriers, and economic challenges, men still highly influence fertility and contraceptive use in many societies in LMICs.54 Consequently, public resources are strained, and opportunities for economic development are hampered.45 Contraceptive uptake can avert millions of preventable deaths, including infant and maternal deaths.Moreover, less contraceptive use, further increases unintended pregnancies resulting in high maternal deaths.46 Henceforth it is considered one of the most reliable FP methods currently available, with very low post-vasectomy pregnancy rates.35 Myths and limited knowledge hinder vasectomy acceptability and its uptake in LMICs. Research on vasectomy has shown that orienting FP services towards men and increasing their knowledge of the method through various media outlets may improve its uptake.46Vasectomy remains one of the least known and least used FP methods in LMICs. The prevalence is less than 1% in LMICs compared to more than 12% in developed countries.55 The analysis also reveals that men generally are reluctant to seek and engage with SRH services for anything other than severe illness.56 Other studies indicate that poor health-seeking behaviour among men requires urgent attention from policymakers. Besides experiencing barriers to care at personal, social, and cultural levels, one possible explanation for men’s low levels of SRH care is a need for greater interest in talking with their healthcare provider about these issues.4The utilisation of SRH services by men is influenced by a complex set of factors related to SRH knowledge; personal; sociocultural norms and beliefs; political will; socioeconomic factors, availability and accessibility of services; quality of the services.41 This abysmal SRH information provision is predominant in urban areas while remaining poor in rural communities.53Sexual and reproductive health information and services are provided through different forms of media, local health pharmacists, public health practitioners, doctors, nurses, and community health workers. However, HIV infection rates, fertility, and unintended pregnancies indicate less frequently used SRH services.18 more studies on men must be conducted, especially in LMICs. Research focusing on men may bring about evidence-based knowledge that will compel policymakers to utilise it when planning to provide SRH services for men. When factors hindering and facilitating men’s SRH service utilisation are known, understood, and dealt with comprehensively and locally, men’s utilisation of SRH services could improve. Subsequently, high morbidity and mortality among men may be averted. The global community must answer the critical question – ‘why do we ignore gender inequity when it impacts the health of boys and men?’. Poor leadership and management are fundamentally linked to failing health systems.59 Therefore, attending to the systemic health barriers is essential to ensure sexual and reproductive justice for all.27This narrative review of the literature revealed a limited number of studies focusing on factors influencing whether male use of SRH services in LMICs. Given the lack of focus on men’s SRH by international programmes such as SDGs 2030 and FP2020,Drawing from the findings of this analysis, we constructed the conceptual model that illustrates factors hindering or facilitating men’s SRH service utilisation . In the This review was confine to LMIC studies only. Therefore, findings may not be generalisable to other settings. The narrative review’s authors also restricted the criteria to include studies published in English because of insufficient resources for translation. English studies may limit how the review’s findings can be transferable to all LMICs. We did not conduct additional study quality assessments or remove studies based on the risk of bias, given that our goal was to describe the relevant studies identified. The predominant focus on VMMC from many studies may result in biased reporting. Narrative reviews often do not meet important criteria to help mitigate bias because they frequently lack explicit criteria for article selection. The paucity of studies focusing on men’s barriers and facilitators to SRH service utilisation may reduce the complexity of the argument. We also included articles that investigated both men and women and those aged 15 years and older, which may have included adolescents. However, authors considered results where authors explicitly separated men’s and women’s results by subheadings. Authors, however, acknowledge that this may introduce bias in the analysis of the results.This review consolidated knowledge about barriers and facilitators influencing men to utilise SRH services. While reviews conducted in the past decades have identified barriers and facilitators to SRH services, our narrative review is the first to comprehensively focus on all SRH services or issues, and men exclusively. The findings from this review have implications for clinical practise and policy.Despite the need for SRH utilisation by men, there is little evidence of successful interventions. Consequently, low SRH utilisation has become a major concern regarding men’s health. Men’s SRH service utilisation must be investigated comprehensively to comprehend men’s problems fully. None of the identified studies comprehensively investigated SRH issues. There is also a need for studies investigating vasectomies and their influence on fertility.54There is a need to explore the utilisation of SRH services among older men as most studies predominantly focus on adolescents and young men, as well as on barriers rather than facilitators to SRH service utilisation. Although SRH includes mental health issues, little is known about mental health issues related to sexual health. The same applies to the impact of physical disabilities and chronic illnesses on sexual well-being. Further research may be warranted in this regard. Governments must increase the awareness and education of the public to fight against myths and misconceptions linked to the utilisation of SRH services and improve healthcare providers’ capacity to engage men better. Furthermost, SRH policymakers and programme managers must avoid frequent changes in SRH prevention programmes as that leads to confusion, despair, and distrust in the health system while discouraging men from engaging with SRH services.


Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis. The developmental process up to the blastocyst stage is a critical period that determines implantation in the uterus3. In early development, spatiotemporal-specific RNA and protein expression is thought to contribute to asymmetric cell division and early cell differentiation4. Single-blastomere transcriptomic analyses have revealed that the molecular symmetry-breaking process already begins as early as the 2-cell stage5. However, much remains unknown about the molecular mechanisms of early embryonic development7. To investigate the role of the temporal and local upregulation of each RNA in early embryogenesis, single-cell-specific RNA delivery methods are useful. In addition, single-cell-specific delivery of small interfering RNA (siRNA) or short hairpin RNA (shRNA) allows us to elucidate the effects of RNA interference (RNAi)-mediated spatiotemporal gene knockdown on early development. Microinjection is capable of single-cell-specific RNA injection, but mechanical damage to the cell is inevitable8. Another methodology that may allow for embryonic single-cell-specific RNA delivery is photo-dependent RNA internalization10.Fertilized eggs undergo repeated cleavages and pass through embryonic stages such as the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages before implantation13. The RNA carrier, TatU1A-Alexa546, is composed of HIV TAT-derived cell-penetrating peptide14, the RNA-binding domain of U1 small nuclear ribonucleoprotein A (U1A)15, and Alexa Fluor 546 (Alexa546) as a photosensitizer. The U1A RNA-binding domain binds to a specific 9-nt RNA sequence. Thus, TatU1A-Alexa546 can deliver RNAs containing the U1A binding sequence. The PCDR procedure is as follows: TatU1A-Alexa546 delivers an RNA to mammalian cells by endocytosis, but the RNA is trapped by endosomes and does not function. Photoirradiation of the photosensitizer generates singlet oxygen, which induces endosomal escape and cytosolic dispersion of the RNA in the irradiated cell16. Thus, via the PCDR method, small RNAs having a U1A binding sequence, such as siRNA, shRNA, and precursor miRNA can be photo-dependently delivered into the cytosol in a time- and region-specific manner19. However, the PCDR method has only been applied to two-dimensionally cultured cells, not to three-dimensional cell assemblies such as embryos. Thus, here we attempted to apply the PCDR method to single-cell-specific cytosolic RNA delivery and the photoinduction of RNAi in mouse embryos.Previously, we developed an RNA delivery method, photoinduced cytosolic dispersion of RNA (PCDR), using photosensitive RNA carriers2. All experimental protocols were approved by the Animal Care and Use Committee of Okayama University . All methods were carried out in accordance with relevant guidelines and regulations of Okayama University. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.Mouse zygotes were collected from superovulated 8- to 12-week-old CD1 female mice. Females were injected with 7.5 IU of pregnant mare serum gonadotropin, followed by 7.5 IU of human chorionic gonadotropin 48 h after pregnant mare serum gonadotropin. Embryos were obtained by mating the superovulated females with CD1 males. For isolation of fertilized zygotes, superovulated females were euthanized by cervical dislocation 20 h post-human chorionic gonadotropin treatment, and zygotes were dissected out of the ampulla in the oviduct. The embryo-cumulus complexes were treated with 300 µg/mL of hyaluronidase to disperse the cumulus cells, washed in M2 medium, and cultured at 37 °C in 5% CO11. The purified TatU1A-C protein (20–30 µM) and Alexa Fluor 546 C5 maleimide  were mixed in T buffer (20 mM HEPES–KOH (pH 7.6), 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 13.8 mM glucose) and incubated at 25 °C for 1 h. The reactant (TatU1A-Alexa546) was purified on a NAP-10 column . The protein concentration of TatU1A-Alexa546 was determined by protein assay CBB solution .RNA carrier proteins with C-terminal Cys residues (TatU1A-C) were purified as described previouslyCA UUG CAC UCC GUC GCG UCC UGG ACG UAG CCU U-3′) and scrambled shRNA  were purchased from JBioS, Japan (the U1A binding sequence is underlined). Fluorescein amidite (FAM)-labeled shRNA (5′-GAU UAU GUC CGG UUA UGU ACA UUG CAC UCC GU CAU AAC CGG ACA UAA UCdT dT-FAM-3′), which is anti-luciferase but nonspecific to mammalian genes, was purchased from Hokkaido System Science, Japan. Before use, all shRNAs were heated at 85 °C for 1 min, followed by annealing by − 1 °C/s to 4 °C.Anti-EGFP shRNA  medium and moved to a 35-mm glass-base dish filled with H-KSOM medium. A single blastomere of the embryo was irradiated via the photobleaching procedure using a laser at a wavelength of 543 nm on a confocal laser scanning microscope . Photobleaching settings were as follows: type of bleaching: Clip Tornado, laser intensity: 20%, repetition: 200 frames. The embryos were then imaged using the confocal laser scanning microscope FLUOVIEW FV-1000.TatU1A-Alexa546 (2 μM), shRNA , and LysoTracker Green  were combined in T buffer containing 0.1% polyvinyl alcohol  and incubated at 37 °C for 10 min. Mouse embryos were treated at 37 °C for 5 min with pronase (Sigma-Aldrich) to remove the zona pellucida. After thoroughly washing nine times with T buffer containing 0.1% PVA, the zona-free embryos were dropped into droplets of the abovementioned TatU1A-Alexa546/shRNA solution (5 µL) in a dish covered with paraffin oil, and incubated at 37 °C under a 5% COT AAT ACG ACT CAC TAT AGG GAC ACA ACT GTG TTT ACT TGC-3′ and 5′-GAT GCT ATT GCT TTA TTT GTA AC-3′ for EGFP, and 5′-CCG GGT AAT ACG ACT CAC TAT AGG TCT ATA TAA GCA GAG CTG G-3′ and 5′-GTT AAC AAC AAC AAT TGC ATT C-3′ for BFP. Capped mRNAs were prepared via in vitro transcription using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) and a polyA addition with the Poly (A) Tailing Kit (Invitrogen) according to the manufacturer's protocols.Template DNAs for EGFP and BFP mRNAs were prepared via PCR using KOD Dash  with 12XCSL-d1EGFP  and pTagBFP-N  vectors as templates. The template DNAs for EGFP and BFP mRNAs were amplified using the following primers (sequence of the T7 promoter is underlined): 5′-CCG GGFor EGFP knockdown experiments, EGFP and BFP mRNA was injected into blastomeres 1 h before the shGFP delivery by PCDR. The solution including 400 ng/µL each of EGFP and BFP mRNAs was loaded into glass micropipettes, and mRNA was injected into an arbitrary blastomere of 4-cell stage embryos using a piezo-driven micropipette unit  and incubated at 37 °C for 1 h. Manipulation was carried out in M2 medium containing 5 mg/mL cytochalasin B (Sigma-Aldrich). The volumes injected typically ranged from 2 to 10 pL, which is 1–5% the volume of the cell.The PCDR method requires the TatU1A-Alexa546/RNA complex to be endosomally entrapped before its photo-dependent cytosolic dispersion. Thus, blastomeres were stained with LysoTracker Green to confirm the subcellular localization of the transfected TatU1A-Alexa546/RNA complex. As a result, LysoTracker Green-stained acidic vesicles such as endosomes co-localized with Alexa546  is not a homologue of EGFP derived from Aequorea victoria, and thus there is no concern of anti-EGFP shRNA misrecognizing BFP. When EGFP mRNA was injected into two cells and only one of the cells was irradiated, EGFP fluorescence disappeared in approximately half of the BFP fluorescent cells after 1 day  used here for photostimulation, red and near-infrared light can also be used in the PCDR strategy. We have previously demonstrated PCDR-mediated, light-dependent RNA delivery using several TatU1A-photosensitizer molecules with photosensitizers such as Alexa633  and DY750 Supplementary Information.


Genomics has transformed our understanding of the genetic architecture of traits and the genetic variation present in plants. Here, we present a review of how RNA-seq can be performed to tackle research challenges addressed by plant sciences. We discuss the importance of experimental design in RNA-seq, including considerations for sampling and replication, to avoid pitfalls and wasted resources. Approaches for processing RNA-seq data include quality control and counting features, and we describe common approaches and variations. Though differential gene expression analysis is the most common analysis of RNA-seq data, we review multiple methods for assessing gene expression, including detecting allele-specific gene expression and building co-expression networks. With the production of more RNA-seq data, strategies for integrating these data into genetic mapping pipelines is of increased interest. Finally, special considerations for RNA-seq analysis and interpretation in plants are needed, due to the high genome complexity common across plants. By incorporating informed decisions throughout an RNA-seq experiment, we can increase the knowledge gained. In 2022, over 5700 articles were identified from the search terms “plant transcriptomics” on the PubMed database, and over 23% of the total data available in the Sequence Read Archive is RNA-seq for algae and land plants . With thTechnologies are rapidly improving to study transcriptomes and becoming more cost effective to be deployed across diverse plants species. While microarrays and RNA-seq are the most common approaches to assess transcriptomes, long-read approaches including Iso-Seq  and NanoFor RNA-seq findings to be impactful, careful consideration in the design and analysis of the experiments are critical. In this review, we highlight considerations for new RNA-seq experiments emphasizing experimental design and best practices for processing RNA-seq data. Gene expression determined from RNA-seq represents an endophenotype, molecular phenotypes intermediate to genotype and organismal traits . Relatin2Thoughtful RNA-seq experimental design is critical for the high-quality data needed to answer complex biological questions. Insufficient experimental design for RNA-seq experiments may lead to makeshift analyses to circumvent inappropriate procedures. These issues have been discussed extensively in human medical literature , but les2.12.1.1k levels, then the study has k treatments. When there is a second factor with j levels applied in combination with factor k, there are k x j treatments, and so on  from raw sequencing reads. The software available are categorized by purpose—quality control, alignment, or quantification—and the genomic information available for the organism. The software described below is commonly used for RNA-seq pipelines; however, this is not a complete list of all software as the tools available are numerous and new software is being developed rapidly .3.1Preprocessing comprises the first step for any sequencing analysis. This stage involves the removal of technical artifacts such as adaptors, PhiX sequence, rRNA sequences, assessing sequence quality and if necessary, quality trimming. Commonly, sequencing centers perform the preprocessing steps of the RNA-seq pipeline, but it is best practice to perform a data quality check in-house. A multitude of tools are available for the preprocessing steps. Quality of raw sequences can be checked via tools such as FastQC  and Multde novo transcriptome analysis, a stringent trimming strategy has also been observed to produce incomplete transcriptome reconstruction . We also detail additional analyses that can be performed to further understand transcriptional profiles.4.1Measuring plant phenotypic plasticity in extreme environments can aid in knowledge to develop plants for future abiotic and biotic environmental conditions arriving with climate change . Using vIncreasingly studies use RNA-seq to understand molecular plant plasticity . The priDifferential expression analysis incorporates a matrix of features’ expression levels and knowledge about the experimental design. Tests for identifying differentially expressed genes (DEGs) rely on contrasting conditions, such as different tissues, genotypes, and conditions. Exploring the up- or downregulation of genes under a stress condition relative to a control condition indicates how a plant combats a stressor and how the stressor harms the plant. For example, in the sorghum lateral root apex, low levels of phosphorus caused major expression changes in the lateral root apices, which correlated with enhanced lateral root growth. Specifically, the low-phosphorus-induced genes encoded proteins with functions in nutrient responses and contribution to phosphorus metabolism . Contras4.2Allele-specific expression (ASE) describes the phenomenon where alleles within a particular genetic feature  have significant differences in their expression levels . The expIn wheat seeds, expression of homeologs and alleles is differentially controlled and consideration of each copy of a gene is relevant. In the endosperm, genes exhibited subgenome dominance in particular functions . Furthercis regulation, where the expression of maize alleles is favored in F1 hybrids between the species. Genes with cis and cis plus trans divergent regulation were enriched among putative targets of selection. In rice, a F1 hybrid of genotypes representing two major subpopulations exhibit enrichment of genes ASE in genomic regions of signatures for domestication or artificial selection  or KOs can be performed by tools like OmicsBox  mapping, genetic loci that determine a trait are examined one-by-one, typically focusing on loci of major effect to find genotypes with a desired allele. In contrast, genomic prediction collectively considers the impact of the entire genotype on a focal trait, especially impactful for traits controlled by many loci of small effect .Within a plant, the genotype is filtered through many levels of endo- or molecular phenotypes to create phenotypes of interest . IntegraRNA-seq supports fine mapping QTL, both by providing additional evidence for the causal gene via differential expression data and by allowing the discovery of new variants in the QTL region, which can be used for marker development . A suitadwarf9 (d9) dominant allele D9-1 had higher abundance of elements like iron, sulfur, and strontium compared to the wild type. D9-1 did not influence cadmium accumulation, as expected by the location of the cadmium GWAS QTL that contained d9. Rather the dwarf allele –D8-mpl – of the paralog d8 identified through co-expression analysis, recapitulated this effect. Co-expression networks can also support extending understanding of the genetic architecture of traits through the examination of epistasis. A maize GWAS for senescence-controlling genes identified putative epistatic interactions among QTL that were independently supported with co-expression data  has prioritized candidate genes . In contion data . Co-exprion data .6Aspects of plant genome biology complicate the use of RNA-seq data, especially relative to the organisms for which many of the bioinformatics tools were developed, though efforts to produce specific tools for polyploid crops are expanding . The gen6.1One feature of plant genomes that can influence the use of RNA-seq is the prevalence of polyploidy . A studyPolyploidy increases computation complexity due to an increase in genome size, relative to a diploid genome and the increased repeat content. The increase in genome size, resulting from the presence of two or more subgenomes, means more memory is required to index and store the reference genome or transcriptome prior to read alignment. As such, analyzing large, polyploid genomes, such as wheat, may be prohibitive to those without expansive compute resources.The degree of similarity among polyploid subgenomes is a driving force for how RNA-seq analyses may need to proceed differently compared to diploid genomes . In the 6.2High levels of repeat content are also problematic from a computational viewpoint to interpret RNA-seq data. Repeat content can refer to both biological repeats  or genomic regions shared between subgenomes. Significant portions of widely grown cereals are comprised of repetitive sequences, excluding homeologs and duplicated genes: wheat-85% , maize-8A. thaliana T-DNA insertion lines at the locations of transposable elements for seedling morphological responses to stresses uncovered functional roles of for over 90% of those tested (EARLY FLOWERING 3 influenced flowering time across A. thaliana accessions (Data loss due to repetitive sequences is problematic as repetitive elements can have important biological functions. For example, screening e tested . Outsidecessions . Inabilicessions  and trancessions  of curre7The ever-increasing need to support a growing global population requires increases in plant productivity and access. New crops and varieties are needed to adapt to challenging abiotic and biotic environmental conditions globally. With the growing affordability and accessibility of RNA-seq, this technology can be leveraged to study any plant species of interest. The most impactful RNA-seq studies are carefully designed and control many sources of unwanted variation. During analysis, much research concludes by examining DEGs between conditions; however, we have outlined additional forms of analysis to extend the usefulness of RNA-seq data, even data already publicly available. The intent of this review is to provide plant-focused guidelines, strategies, and examples for supporting new users of RNA-seq and inspiration for new applications by established researchers.RNU, FHC, JLi, GLR, KF, JPC, and JLa drafted and finalized themanuscript. All authors contributed to the article and approved the submitted version.


Dysregulation of mRNA processing results in diseases such as cancer. Although RNA editing technologies attract attention as gene therapy for repairing aberrant mRNA, substantial sequence defects arising from mis-splicing cannot be corrected by existing techniques using adenosine deaminase acting on RNA (ADAR) due to the limitation of adenosine-to-inosine point conversion. Here, we report an RNA editing technology called “RNA overwriting” that overwrites the sequence downstream of a designated site on the target RNA by utilizing the RNA-dependent RNA polymerase (RdRp) of the influenza A virus. To enable RNA overwriting within living cells, we developed a modified RdRp by introducing H357A and E361A mutations in the polymerase basic 2 of RdRp and fusing the C-terminus with catalytically inactive Cas13b (dCas13b). The modified RdRp knocked down 46% of the target mRNA and further overwrote 21% of the mRNA. RNA overwriting is a versatile editing technique that can perform various modifications, including addition, deletion, and mutation introduction, and thus allow for repair of the aberrant mRNA produced by dysregulation of mRNA processing, such as mis-splicing. Dysregulation of mRNA processing can lead to various diseases, including cancer, which are called RNA diseases ,2. RecenThe wild-type RdRp performs editing of viral mRNA by targeting host cell mRNAs through a cap-snatching mechanism a. TherefPreviously, the interaction system between the Box B sequence within the gRNA and the λn  was usedWe performed RNA overwriting in HEK293T cells using RNP containing either PA-dCas13b or PB2-dCas13b. To estimate cleavage and overwriting efficiency from fluorescence intensity, we constructed a fluorescent reporter assay system that overwrites EGFP mRNA with mScarlet-I mRNA a. The seWe performed RNA overwriting using the AG site within the open reading frame (ORF) as the editing start point a. We des2.A 293T human embryonic kidney (HEK 293T) was cultured with Dulbecco modified Eagle medium  supplemented with 10% fetal calf serum , 100 units/mL penicillin, and 100 mg/mL streptomycin (Fujifilm) at 37 °C in 5% COI) and the mouse RNA polymerase I terminator (tI) sequence into pcDNA3, truncating the CMV promoter and BGH polyadenylation signal sequence. pPolI-mScarlet was constructed by inserting the mScarlet-I sequence between pI and tI of the pPolI plasmid in the negative-sense orientation  were subcloned into pcDNA3 plasmid and designed pcDNA3-PA, -PB1, -PB2, or -NP. The plasmid of PA, PB1, or PB2 fused with the dCas13b at N- or C-terminus was obtained by cloning the sequence of dCas13b into pcDNA3-PA, PB1, and PB2. The plasmids for the PB2 mutant were prepared using a PCR-based site-directed mutagenesis method. The pPolI plasmid was constructed by inserting the synthetic human RNA polymerase I promoter  plasmids was co-transfected with 0.6 μg of pcDNA-NP and 0.3 μg of pPolI-mScarlet plasmids using Lipofectamine LTX  and Opti-MEM (Thermo Fisher Science) according to the manufacturer’s instructions. At 48 h post-transfection (hpt), red fluorescence images were taken using a fluorescence microscope , and cells were lysed in 100 μL of lysis buffer  at 4 °C for 60 min. The lysates were analyzed using a fluorescence microplate reader .The HEK 293T cells were seeded one day prior to transfection on a 3.5 cm glass bottom or normal culture dish with 5 × 106 cells/10 mL. A total of 2.7 μg each of the pcDNA-PA (-dCas13b-PA or -PA-dCas13b or PA-strep), -PB1 (-dCas13b-PB1 or -PB1-dCas13b), and -PB2 (-dCas13b-PB2 or -PB2-dCas13b or PB2-mutant) plasmids was co-transfected with 2.5 μg of pcDNA-NP and 1.3 μg of pPolI-mScarlet plasmids in HEK 293T cells using Lipofectamine LTX and Opti-MEM. The sequence of twin-strep-tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) was inserted into dCas13b-PA (or PA-dCas13b), dCas13b-PB1 (or PB1-dCas13b), and dCas13b-PB2 (or PB2-dCas13b) before (or after) the dCas13b. At 48 hpt, cells were lysed in 500 μL of lysis buffer at 4 °C for 60 min. To the lysate in 2.5 mL of binding buffer , Strep-Tactin XT 4Flow (IBA) (50 μL) was added and incubated at 4 °C for 3 h. The Strep-Tactin XT 4Flow was then washed three times with 3 mL of wash buffer . The RNP complex was released from the Strep-Tactin XT 4Flow by elution buffer  at 4 °C for 1 h and analyzed by 7.5% SDS-PAGE, followed by silver staining.The HEK 293T cells were seeded one day prior to transfection on a 10 cm culture dish with 4 × 10Western blot analysis for NP was performed by electrophoretic transfer of the 7.5% polyacrylamide gel to a PVDF membrane (ATTO) in blotting buffer  for 1 h. The membrane was then incubated for 1 h with blocking buffer . After washing with TBS, the membrane was incubated with anti-NP mouse monoclonal antibody  diluted 1:1000 in TBS containing 0.1% Tween-20 and 5% BSA. Then the membrane was washed with TBS, followed by incubation at 4 °C overnight with HRP-conjugated goat anti-mouse IgG secondary antibody  diluted 1:5000 in TBS containing 0.1% Tween-20 and 5% skim milk. After washing with TBS, blots were developed via staining solution .6 cells/10 mL. A total of 2.7 μg each of the pcDNA-PA, -PB1, and -PB2 (or PB2-mutant) plasmids was co-transfected with 2.5 μg of pcDNA-NP and 1.3 μg of pPolI-mScarlet plasmids in HEK 293T cells using Lipofectamine LTX and Opti-MEM. At 48 hpt, the total RNA was extracted using TRI Reagent . The mScarlet-I mRNA and template RNA were reverse transcribed using FAM-labeled primer . The products were analyzed by 15% denaturing PAGE containing 8 M urea in Tris-glysine buffer.The HEK 293T cells were seeded one day prior to transfection on a 10 cm culture dish with 4 × 105 cells/2 mL. A total of 0.2 μg each of the pcDNA-PA (or -PA-dCas13b), -PB1, -PB2 (or -PB2-dCas13b), and pcDNA-NP plasmids and 0.1 μg of the pPolI-mScarlet plasmid were co-transfected with 30 ng of pcDNA-target plasmids in the presence or absence of 3.8 μg of phU6-gRNA plasmids in HEK 293T cells using Lipofectamine LTX and Opti-MEM. At 48 hpt, green and red fluorescence images were taken using a fluorescence microscope, and cells were lysed in 100 μL of lysis buffer at 4 °C for 60 min. The lysates were analyzed using a fluorescence microplate reader. To inhibit the endonuclease activity of the PA subunit, the inhibitor L742001 (Sigma-Aldrich) (21.5 μM) was added to the culture medium. For preparation of the sequencing sample, the total RNA was extracted using TRI Reagent. The extracted RNA was reverse transcribed to cDNA using RT primer (5′-CTTGTACAGCTCGTCCATGC-3′), followed by amplification by PCR using primers . The product was directly sequenced. The relative overwriting efficiency was calculated in comparison to the case when RNA overwriting was performed using plasmids for expression of mScarlet-I mRNA instead of target EGFP mRNA.The HEK 293T cells were seeded one day prior to transfection on a 3.5 cm glass bottom or normal culture dish with 5 × 10In summary, we performed two optimizations to generate a modified RdRp to conduct RNA overwriting within living cells. First, we searched for a mutant of PB2 that does not bind to the 5′-cap of mRNA and is capable of forming RNP for transcription and replication. We found an optimal mutant containing H357A and E361A. Second, we optimized the fusion site for dCas13b. Because dCas13b is large, it inhibited RNP formation when fused to the N-terminus of PA, PB1, and PB2 or the C-terminus of PB1. However, when dCas13b was fused to the C-terminus of PA or PB2, RNP was formed, with its activity maintained at 75–90% compared to the wild type. Thus, we demonstrated the in-cell overwriting of EGFP mRNA with mScarlet-I mRNA using a modified RdRp containing either PA-dCas13b or PB2-dCas13b. When the editing was initiated at the AG site 12 nucleotides downstream of the double-stranded region with gRNA using the PB2-dCas13b-containing RNP, the highest editing efficiency was observed, cleaving 46% of EGFP mRNA and overwriting 21% with mScarlet-I mRNA. Finally, RNA overwriting was performed with the editing start point within the ORF. While the cleavage efficiency of the target mRNA was 51%, the overwriting efficiency was 5% due to the change in the intramolecular double-stranded region of the template RNA from the vRNA sequence to that of mScarlet-I. Moreover, mutations were introduced into the overwritten sequence because the RNA motif of vRNA was required at the 3′-end of the template RNA. Compared to the RNA editing technology using ADAR, which can only perform A-to-I point editing, RNA overwriting is a versatile editing technique that can perform various modifications, including addition, deletion, and mutation introduction. Therefore, our technology is expected to be applicable in gene therapy to treat RNA diseases caused by dysregulation of mRNA processing, such as mis-splicing. For instance, cancer cells often contain abnormal mRNA of p53, produced by mis-splicing of the TP53 tumor suppressor gene . Our tec


The functions of non-coding RNAs usually depend on their 3D structures. Therefore, comparing RNA 3D structures is critical in analyzing their functions. We noticed an interesting phenomenon that two non-coding RNAs may share similar substructures when rotating their sequence order. To the best of our knowledge, no existing RNA 3D structural alignment tools can detect this type of matching. In this article, we defined the RNA 3D structure circular matching problem and developed a software tool named CircularSTAR3D to solve this problem. CircularSTAR3D first uses the conserved stacks (consecutive base pairs with similar 3D structures) in the input RNAs to identify the circular matched internal loops and multiloops. Then it performs a local extension iteratively to obtain the whole circular matched substructures. The computational experiments conducted on a non-redundant RNA structure dataset show that circular matching is ubiquitous. Furthermore, we demonstrated the utility of CircularSTAR3D by detecting the conserved substructures missed by regular alignment tools, including structural motifs and conserved structures between riboswitches and ribozymes from different classes. We anticipate CircularSTAR3D to be a valuable supplement to the existing RNA 3D structural analysis techniques.  The 3D structure of non-coding RNAs is one of the primary determinants of their functions. Comparing their structures can help researchers understand their functional and evolutionary relationships. A variety of RNA 3D alignment algorithms have been developed. One of the popular strategies is to represent RNA 3D structures by one-dimensional sequences consisting of structural alphabets , dihedracircular match if their 3D structures can be aligned when the sequence order is rotated in one of the RNAs. The terms circular match, circular alignment and rotated match are used interchangeably in this study. The circular matching problem for RNA structural motifs has been solved by aligning loop regions independently by using motif alignment tools such as RNAMotifScan and concatenating them in different ways , nine instances of kink-turn motifs have been identified  . For exaentified ,23. By uentified  as the qentified . Using tH. marismortui 23S rRNA . The instances in three other rRNAs are shown in Supplementary Figure S3. In the alignments of these motif instances, only two of the three conserved stacks are detected and used as anchors. However, CircularSTAR3D still includes the whole multiloop in the alignment, illustrating the robustness of our method. Compared with the alignments generated by RNAMotifScan  on the BGSU RNA site by using ‘riboswitch’ as the search filter . The dathia coli  and a PRathranii . This ale ATPase . The PRPRibozymes are RNA molecules that have catalytic functions. Circular matching may help researchers study naturally occurring ribozymes as well as design synthetic ribozymes. A circularly permutated ribozyme was designed to study the structure and function of a lariat capping ribozyme . We usedTo comprehensively identify the circular matches in ribozymes, we downloaded a set of ribozyme structures in the ‘Representative Sets of RNA 3D Structures’ list (version 3.141) , which cIn this study, we presented a novel computational tool for circular matching, named CircularSTAR3D. It can detect the circular matches between the input RNA structures. CircularSTAR3D first identifies the rotated matched internal loops and multiloops in the input RNAs. Then it extends from all the conserved stack pairs that close these loops. This ‘two-step’ strategy is derived based on two observations. First, internal loops and multiloops can have circular matches when concatenating the loop regions in different ways. The hairpin loops only have one loop region and do not have circular alignment. Second, a pair of rotated matched loops are closed by at least one rotated matched stack pair. By integrating these two properties into the design, we developed CircularSTAR3D as a new ‘stack-based’ RNA 3D structure alignment algorithm. By using the aligned stacks as anchors, CircularSTAR3D avoids the all-to-all loop matching and enumerating different ways in which concatenation can occur. In most cases, the current implementation can find all rotated regions between two RNA 3D structures in 10 s on a modern desktop. We provide CircularSTAR3D and LocalSTAR3D in one software package so that users can generate both rotated and non-rotated matches.Besides the alignment examples we have shown in the Results, a potential application is to integrate it into RNA clustering pipelines along with STAR3D and LocalSTAR3D. CircularSTAR3D outputs an alignment score to sort the aligned regions in a pair of RNA 3D structures. For clustering applications, we need to compare the alignments from different pairs of input RNAs, and a size-independent score should be used for that purpose, such as RMscore  and TM-shttps://github.com/ucfcbb/CircularSTAR3D and https://doi.org/10.5281/zenodo.7719548.The CircularSTAR3D stack-based RNA 3D structural alignment tool is available at gkad222_Supplemental_FilesClick here for additional data file.


Lox gene expression and crosslinking activity were increased. Overall, our results suggest that CD11b integrin regulates cartilage calcification through reduced MV release, apoptosis, LOX activity, and matrix crosslinking. As such, CD11b activation might be a key pathway for maintaining cartilage integrity.Pathological cartilage calcification is a hallmark feature of osteoarthritis, a common degenerative joint disease, characterized by cartilage damage, progressively causing pain and loss of movement. The integrin subunit CD11b was shown to play a protective role against cartilage calcification in a mouse model of surgery-induced OA. Here, we investigated the possible mechanism by which CD11b deficiency could favor cartilage calcification by using naïve mice. First, we found by transmission electron microscopy (TEM) that CD11b KO cartilage from young mice presented early calcification spots compared with WT. CD11b KO cartilage from old mice showed progression of calcification areas. Mechanistically, we found more calcification-competent matrix vesicles and more apoptosis in both cartilage and chondrocytes isolated from CD11b-deficient mice. Additionally, the extracellular matrix from cartilage lacking the integrin was dysregulated with increased collagen fibrils with smaller diameters. Moreover, we revealed by TEM that CD11b KO cartilage had increased expression of lysyl oxidase (LOX), the enzyme that catalyzes matrix crosslinks. We confirmed this in murine primary CD11b KO chondrocytes, where  Another mechanism of crystal formation is apoptosis, during which apoptotic bodies of 1000–5000 nm [Pathological calcification (PC) of articular cartilage is a hallmark of osteoarthritis (OA), a degenerative disease of the joints that affects globally over 300 million people  leading –5000 nm  are rele–5000 nm ; up to 1Mβ2 or Mac-1 for macrophage antigen 1), a member of the β2 family of leukocyte adhesion receptors, mostly studied for its anti- and pro-inflammatory capabilities in autoimmune diseases [A novel integrin studied in OA cartilage is CD11b/CD18 , while the same genes were not modulated in CD11b KO cells cultured in the same conditions (Lox gene was downregulated in WT but not in CD11b KO chondrocytes cultured on less crosslinked matrices (BAPN-treated)  were used. Animals were kept in a temperature-controlled and ventilated rack with a 12:12 h light cycle and with access to water and food ad libitum.2 inhalation. Intact right knees were dissected and fixed overnight at 4 °C in 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde in 100 mM phosphate buffer (PBS) with pH 7.4 for morphological TEM analysis. Left knees were fixed overnight at 4 °C 2% (v/v) formaldehyde and 0.25% (v/v) glutaraldehyde in 100 mM phosphate buffer (PBS) with pH 7.4 for immuno-gold electron microscopic analysis. From the day after, samples were stored in 100 mM PBS at 4 °C.WT and CD11b KO female mice (n = 4 for each genotype) were sacrificed at 13 weeks old via CO2 inhalation. Intact right knees were dissected and fixed overnight as described above for morphological TEM analysis.WT and CD11b KO female mice (n = 4 for each genotype) were sacrificed at 77 weeks old via COv/v) osmium tetroxide and 1% (w/v) potassium hexacyanoferrate (III) in 0.1 M cacodylate buffer for 2 h at 4 °C, followed by washing with distilled water. After dehydration in an ascending ethanol series from 30 to 100%, specimens were incubated two times in propylene oxide each for 15 min and embedded in Epon using flat embedding molds. Tissue samples used for immuno-gold electron microscopic analysis were rinsed in PBS after fixation, dehydrated in ethanol up to 70%, and embedded in LR White embedding medium  using beem capsules. Ultrathin sections were cut with an ultramicrotome, collected on copper grids, and negatively stained with 2% uranyl acetate for 15 min. For immuno-gold electron microscopy, ultrathin sections were incubated with 100 mM glycine in PBS for 2 min, washed with PBS, and blocked with 2% (w/v) BSA and 1% normal goat serum in PBS. For immuno-labeling, grids were incubated for 1 h at room temperature on drops of primary antibodies (ALP (GTX100817) from GenTex , 1:25; ANX V (PA5-78784) from Invitrogen , 1:100; and LOX (E-19) from Santa Cruz Biotechnology , 1:100 diluted in PBS containing 1% (v/v) BSA-c  and 0.025% (v/v) Tween 20. After washing with the same solution, ultrathin sections were incubated with secondary antibodies (diluted 1:20) conjugated to 18 nm gold particles. After washing with distilled water, ultrathin sections were negatively stained with 2% (w/v) uranyl acetate for 10 min. Electron micrographs were taken at 60 kV with a Phillips EM-410 electron microscope using imaging plates . For quantification of the immuno-gold labeling, the number of gold particles was counted on several fields of EM images of WT and CD11b KO animals. The determinations of the areas of mineralization, matrix vesicles, immuno-gold labeled proteins, and fibril diameters were performed by using ImageJ 1.52q  [Afterward, cartilage was carefully dissected from the knees. Tissue samples used for morphological analysis were postfixed in 0.5% , in the presence or absence of the pan-LOX(L) inhibitor β-aminoproprionitrile  to form matrices with various degrees of crosslinks . Cells wg for 10 min to remove dead cells and debris. Then, a second centrifugation at 2000× g for 10 min was performed to pellet apoptotic bodies (1000–5000 nm). A third centrifugation at 10,000× g for 30 min allowed us to pellet and remove larger microvesicles (>100–1000 nm). The final centrifugation at 100,000× g for 70 min was done to pellet matrix vesicles. Pelleted MVs were resuspended in PBS, and RIPA buffer was added to disrupt the phospholipidic layer in order to measure calcium content with QuantiChromTM Calcium Kit .Extracellular vesicles of 30–150 nm diameter  were isolated from chondrocytes supernatants by serial ultracentrifugations at 4 °C  using OpTM, Waltham, MA, USA) and 7-amino-actinomycin staining (7AAD) . ANX V/7AAD double staining is a convenient way to discriminate early apoptosis from late apoptosis and necrosis. In particular, during early chondrocyte apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane, and it binds to the transmembrane protein ANX V, whose on-surface staining can be therefore used to detect apoptosis [Chondrocytes were detached from culture plates by use of non-enzymatic cell dissociation buffer (5 mM EDTA in PBS). Cells were then suspended in FACS buffer  and labeled with AnnexinV-PE  with INSPIRE software , then evaluated in IDEAS software  version 6.0.Analysis was performed in LSRII cytometer using FACS Diva6  and FlowJoX software , version 9, for data processing. Imaging flow cytometry was performed using the ImageStreamTM. Real time quantitative PCR on specified gene primers . Results were normalized on Gapdh as a housekeeping gene, and delta-delta CT was calculated on control samples.Cells were homogenized using TRIzol  (500 µL for 1 million cells), and RNA extraction was performed using RNA Clean & Concentrator5-Zymoresearch. Complementary DNA was synthesized from RNA with Superscript II-Invitrogen primers  was perfLOX activity was measured in supernatants of WT and CD11b KO chondrocytes cultured in DMEM without phenol red for 48 h by use of PromoKine Lysyl Oxidase Activity Assay Kit from PromoCell .WT and CD11b KO chondrocytes were cultured in T75 flasks for 3 weeks in DMEM low glucose . Then, cell layers were washed with PBS, scraped, and stored for collagen analysis at −20 °C. Analysis of crosslinks (dihydroxylysinonorleucine (DHLNL), hydroxylysinonorleucine (HLNL), and histidinohydroxymerodesmosine (HHMD)) was performed as described previously ,45. The t-test or one-way or two-way Anova, depending on the type of data. Statistical significances were shown as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.All data were calculated by mean ± standard deviation of triplicates or more. Graphs were made in GraphPad Prism software , version 9. Variation amongst genotypes and experimental conditions were calculated using Student’s


The ribosome is one of the largest complexes in the cell. Adding to its complexity are more than 200 RNA modification sites present on ribosomal RNAs (rRNAs) in a single human ribosome. These modifications occur in functionally important regions of the rRNA molecule, and they are vital for ribosome function and proper gene expression. Until recent technological advancements, the study of rRNA modifications and their profiles has been extremely laborious, leaving many questions unanswered. Small nucleolar RNAs (snoRNAs) are non-coding RNAs that facilitate and dictate the specificity of rRNA modification deposition, making them an attractive target for ribosome modulation. Here, we propose that through the mapping of rRNA modification profiles, we can identify cell-specific modifications with high therapeutic potential. We also describe the challenges of achieving the targeting specificity needed to implement snoRNAs as therapeutic targets in cancers. DNA and histone modifications (epigenomics) have an established role in regulating gene expression and disease onset ,2,3. HowWith 80 proteins and 4 RNA molecules, the ribosome is one of the largest complexes in the cell . Adding Together, 2′OMe and Ψ comprise more than 95% of modified rRNA nucleotides, with more than 110 2′OMe and ~100 Ψ modified residues in a single ribosome ,22,23,24Even though many RNA modifications have been known since the 1950s, due to technological limitations, their investigation has thus far been extremely laborious and mostly confined to a single modified site at a time. This hindered our understanding of their function. Moreover, the lack of genetic tools for research in vertebrates and/or mammalian models has restricted our capacity to explore their physiological function and relevance to human disease. In the past decade, we have witnessed a boom in technological advancements, making it possible to detect RNA modifications transcriptome-wide and with single nucleotide resolution. This technological leap enables us to revisit the contribution of rRNA 2′OMe and Ψ in human disease to reassess the therapeutic potential of snoRNAs.Indeed, in recent years, aberrant snoRNA expression has been described in various malignancies. The description of this body of work has been extensively reviewed by others ,26,27. HSnoRNAs are ubiquitous and highly abundant RNAs ,28. So, Recent publications have demonstrated that some rRNA modifications occur in sub-stoichiometric levels ,29. ThisThese cancer-specific modification profiles, once identified, will lead to the subsequent snoRNAs, which are crucial for the malignant cell. Those snoRNAs would have the highest potential as candidates for therapeutic targeting due to their ability to provide cancer specificity, and so their targeting will mostly affect cancer cells and not healthy, by standard, cells.Recent works have already uncovered a few such snoRNA-dependent modification sites crucial for the maintenance of cancers. Zhou et al. revealed that rRNA 2′OMe modulates protein translation, affecting leukemia stem cell (LSC) phenotypes and propagation . Their wAdditionally, Pauli and Liu et al. uncovered another single methylation site important for the proliferation of AML cell lines . They peHerein, we focus on leukemia; however, snoRNAs have been found to be implicated in additional cancers ,32. For The above-mentioned examples demonstrate the central role of even a single modification site on rRNAs in regulating malignancies. The need of the malignant cell to tailor its ribosome and achieve a high proliferative rate becomes a liability as it generates specificity. Thus, the identification of tumor-specific modification profiles may expose new therapeutic targets and pose as a promising avenue for cancer therapy.Several techniques have been developed to target cellular RNAs. Here, we discuss two.ASOs are single-stranded deoxy-oligonucleotides, usually ~20 nt in length, which are designed to bind specifically to the target RNA and modulate its abundance. ASOs act through several mechanisms of action. Regarding the regulation of snoRNA abundance, we bring forth two such mechanisms.Ribonuclease H1, or RNase H1, resides in nuclei ,38 and pMost snoRNAs are encoded from the introns of host genes . The hosAn interesting and promising alternative to sequence-specific targeting is structure-specific small molecules that bind to structures in the RNA molecule. Small molecules can be designed to lead to the degradation of their target, thus modulating their targets’ function. Small molecules are attractive therapeutic agents as they have low molecular weight and are less charged. These traits facilitate improved delivery efficiency compared with ASOs. Importantly, small molecules have been shown to be bioactive and tolerated in multiple models ,50. ImpoTo identify potential small molecules in a high-throughput approach, RNA motif libraries are used to screen for small molecules that bind to a certain motif with high specificity. As these libraries are modular, they enable diverse experimental designs where one can either screen for the binding of many motifs or, alternatively, focus on specific motif families. Various mechanisms of action have been demonstrated by small molecules; for example, direct binding of the target RNA and its neutralization. Other small molecules have been designed to recruit endogenous nucleases, leading to the degradation of the target RNA ,52. The Thanks to technological developments and the rise of the non-coding RNA field, snoRNAs are now appreciated as facilitators of many molecular processes. These include ribogenesis, translation regulation by rRNA modifications or miRNAs, splicing, and more ,54. BecaThe growing appreciation of cell-type-specific rRNA modification profiles ,15,29 en


Liver metastasis is the leading cause of death in patients with colorectal cancer (CRC). Emerge evidence suggests that circular RNA (circRNA) is a pivotal player in cancer progression. However, its role in CRC liver metastasis remains largely unknown.Circ-YAP expression was detected by qRT-PCR and in situ hybridization. The function of circ-YAP was tested by wound healing, transwell and CCK-8 assays. RNA immunoprecipitation, pull-down, luciferase reporter, chromatin immunoprecipitation assays were used to investigate the mechanism underlying circ-YAP promoting CRC liver metastasis. CRC liver metastasis animal model was established to assess the effect of circ-YAP in vivo.6-methyladenosine (m6A) modification orchestrated efficient initiation of circ-YAP translation, requiring m6A reader YTHDF3 and eIF4G2 translation initiation complex. Intriguingly, circ-YAP was transcriptionally enhanced by YAP/TEAD complex, thus forming a positive regulatory feed-forward loop.Circ-YAP was notably upregulated in CRC with liver metastasis, which was associated with dismal prognosis. Circ-YAP promoted CRC cell migration and invasion in vitro, and facilitated liver metastasis in patient-derived xenografts (PDX) models in vivo. Mechanistically, circ-YAP encoded a novel truncated protein containing 220 amino acids, termed as YAP-220aa, which competitively bound to LATS1, resulting in YAP dephosphorylation and nuclear translocation, thereby activating a cohort of metastasis-promoting genes. Importantly, NOur findings reveal a previously uncharacterized oncoprotein encoded by circ-YAP, implying a promising biomarker and therapeutic target for CRC patients with liver metastasis.The online version contains supplementary material available at 10.1186/s12943-023-01848-1. Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, with the incidence and mortality ranking the third and second among all malignant tumors, respectively . Tumor min silico approaches have identified that circRNA is highly conserved and widely expressed in a disease-, tissue- or cell-specific pattern [6A) modification [6A is the most abundant internal modification of eukaryotic RNA, which is dynamically reversible and catalyzed by m6A writer (METTL3/14 and WTAP), removed by m6A eraser (FTO and ALKBH5) and recognized by m6A reader (YTHDF1/2/3 and YTHDC1/2) [Circular RNA (circRNA) is a special class of endogenous RNA molecules, with a covalently closed loop structure, without the traditional 5’-end “cap structure” and 3’-end poly A tail . It is g pattern . Increas pattern , 8. The  pattern , the mos pattern , 11. Mor pattern . Interes pattern , 14. For pattern . In addi pattern and hepat pattern , respectfication . m6A is THDC1/2) . To date6A-dependent manner. Further, we found that YAP-220aa activated YAP signaling via preventing LATS1-mediated YAP phosphorylation and cytoplasmic retention.In this study, we identified a circRNA, circ-YAP, as a driver of CRC liver metastasis. Circ-YAP contains a 220-aa open reading frame (ORF) and encodes a novel YAP protein isoform, termed as YAP-220aa, in an mAll CRC tissues included in this study were collected from Sun Yat-sen University Cancer Center, including fresh frozen  and paraffin embedded  tissues. Written informed consent was obtained from each subject. This study was approved by the Institutional Ethical Review Boards of Sun Yat-sen University Cancer Center (SL-B2022-276-02). The normal FHC cells (CRL-1831) and CRC cells including HT-29 (HTB-38), SW480 (CCL-228), DLD1 (CCL-221), HCT116 (CCL-247), SW620 (CCL-227) and LoVo (CCL-229) were obtained from ATCC. The above cells were cultured in DMEM medium with 10% fetal bovine serum (FBS). Besides, TC71 PDX cells were obtained from XENTECH (No. XTM-233_CXT-399/R5700), and cultured in advanced DMEM/F12 medium supplemented with 8% FBS, 1% antibiotics and 1% glutamin. All cells were periodically tested for mycoplasma contamination.Total RNA was isolated using Trizol reagent , then, 1 μg RNA was reverse transcribed into cDNA using PrimeScript RT Enzyme . RNA amplification and quantification were carried out with TB Green Premix Ex Taq II Kit (Takara Bio). The specificity of all primers was verified by the melt curve and the sequences are listed in Table The FAM-labeled probe targeting the junction site of circ-YAP was designed and synthesized by GenePharma , followed by hybridization using the FISH Kit according to the manufacturer’s instructions (GenePharma). For ISH assay, the paraffin-embedded tissues were digested with proteinase K, and incubated with 5’-digoxin-labeled probe against circ-YAP junction site at 55 °C overnight. After incubation with anti-digoxin antibody  at 4 °C overnight, the slides were stained with NBT/BCIP reagent. For IHC staining, the anti-YAP-220aa (produced by GenScript) and anti-YAP  antibodies were used with the dilution ratio of 1:50 and 1:400, respectively. The protein signals were visualized by DAB solution. The semi-quantitative analysis of ISH/IHC staining was conducted using H-score method as previously described [6A motif was conducted using Q5 Site-directed Mutagenesis Kit  according to the manufacturer’s instructions. All constructs were confirmed by Sanger sequencing. Cell transfection was carried out using Lipofectamine 3000 (Invitrogen) and the transfection efficiency was tested by qRT-PCR or western blot assays.To silence circ-YAP, the CRISPR/Cas13d system was used . In brieCell migration was tested by wound healing assay. Briefly, CRC cells were plated into 6-well plates, and the scratches were generated using a sterile pipette tip. Then, cells were cultured in DMEM medium without FBS. After 24 h, the migration distance was recorded. For detection of cell invasion, the upper chamber with an 8 mm pore size filter  was used and the bottom chamber was filled with 600μL DMEM complete medium. After 24 h, the invaded cells were stained by crystal violet. For CCK-8 assay, CRC cells were plated into 96-well plates, followed by incubation with 10μL CCK-8 reagent  at 37 °C for 2 h. The absorbance at 450 nm of each well was recorded with a microplate reader.6 cell suspension into the colonic subserosa of NOD/SCID mice. After 10 weeks, mice were euthanized, and liver tissues were carefully dissected out to detect for metastatic lesions. For the experimental liver metastasis model, 20μL 1 × 106 TC71 PDX cells suspended in PBS were slowly injected into spleen, after 4 weeks, the number of liver metastatic nodule in each group was counted. Liver metastasis burden was defined as the number of metastatic nodules multiplied by the diameter of the metastatic lesions. All procedures for animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (SYSU-IACUC-2021-000653).For the spontaneous liver metastasis model, TC71 PDX cells with or without circ-YAP knockdown were harvested by trypsinization and washed three times with cold PBS, followed by orthotopically injection of 50μL 3 × 10Cells were washed by cold PBS and lysed by RIPA buffer supplemented with 1×protease inhibitor cocktail (Roche). The concentration of protein was detected by Pierce™ BCA Protein Quantification Kit (Invitrogen). Equal amount of protein was loaded onto 8-10% SDS-PAGE gel and transferred onto PVDF membrane. Then, the membrane was incubated with appropriate primary and secondary antibodies, and visualized using Pierce™ ECL Western solution (Invitrogen). For Co-IP assay, cell lysates were pre-cleared by incubating with 20μL protein A/G agarose . After that, the supernatant was incubated with appropriate primary antibody at 4 °C for 3 h, followed by incubation with 40μL protein A/G agarose at 4 °C for 30 min. The enriched proteins were separated by SDS-PAGE gel and analyzed by western blot. The primary antibodies used in this study were as follows: anti-Flag , anti-YAP-220aa (provided by Genescript), anti-METTL3 , anti-YTHDF1 , anti-YTHDF2 , anti-YTHDF3 , anti-eIF4G2 , anti-eIF4A , anti-eIF4B , anti-LATS1 , anti-YAP , anti-14-3-3 , p-YAP (S127) , anti-TEAD1 , anti-Tubulin , anti-CDX2 , anti-GAPDH  and anti-Histone H3 .CRC cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked using 5% BSA solution for 30 min at room temperature.Then, cells were incubated with anti-Flag, anti-YAP-220aa and anti-YAP antibodies overnight at 4 °C. After incubating with fluorescein-conjugated secondary antibody for 1 h, the fluorescence signal was observed using a fluorescence microscope. Cell nucleus was stained with DAPI solution.6A Kit  was used. Total RNA was fragmented into 100nt or less for 5 min at 70 °C, followed by incubation with anti-m6A antibody  and Magna Protein A/G Magnetic Beads overnight at 4 °C. The enriched RNA was eluted by m6A 5′-monophosphate sodium salt, followed by RNA extraction and qRT-PCR analysis.RIP assay was conducted using the Magna RIP Kit  as per the manufacturer’s protocols. In short, the magnetic beads pre-coated with anti-YTHDF3 or anti-IgG (Millipore) were incubated with CRC cell lysates at 4 °C for 12 h. After protein digestion by proteinase K, the enriched RNA was extracted by Trizol reagent, followed by qRT-PCR analysis of circ-YAP level. For meRIP assay, the Magna MeRIP™ mFor in vivo pull-down assay, the biotin-labeled probe targeting circ-YAP junction site was synthesized and incubated with CRC cell lysates at 4 °C for 5 h, followed by incubation with the Streptavidin Magnetic Beads (Invitrogen) at 4 °C for 1 h. Then, the enriched proteins were eluted for western blot analysis. For in vitro pull-down assay, the linear circ-YAP was in vitro transcribed using the T7 Transcription Kit (Invitrogen) and labeled with biotin using Biotin RNA Labeling Mix (Roche), followed by circularization using T4 RNA ligase I. The above synthesized circ-YAP was incubated with recombinant human YTHDF3 protein  in binding buffer  at 4 °C for 1 h, followed by incubation with the Streptavidin Magnetic Beads (Invitrogen) and western blot analysis.Cells were incubated with 5,6-dichlor-obenzimidazole 1-β-D-ribofuranoside  to block transcription. After DRB release, the newly transcribed RNA was labeled with 4-thiouridine . Total RNA was extracted using Trizol reagent, followed by biotinylation and pull-down with the Streptavidin Magnetic Beads (Invitrogen). The nascent RNA was serially washed and subjected for qRT-PCR analysis of circ-YAP level.For detecting YAP transcription activity, cells with circ-YAP silencing or overexpression were transfected with YAP luciferase reporter  using Lipofectamine 3000 (Invitrogen). After 48 h of transfection, the relative luciferase activity was detected by the dual-luciferase reporter system (Promega) according to the manufacturer’s instructions. For analysis of circ-YAP promoter activity, the full-length or truncated circ-YAP promoter was inserted into pGL3-basic vector (Promega), followed by co-transfection with pRL-TK and YAP-5SA vectors into cells using Lipofectamine 3000 (Invitrogen). The relative luciferase activity was tested as mentioned above. Each group was run in triplicate in 48-well plates.ChIP assay was carried out using the commercialized SimpleChIP® Plus Sonication Chromatin IP Kit  based on the manufacturer’s protocols. The used ChIP-grade antibodies are as following: anti-YAP , anti-PoI II  and anti-p-PoI II (S5) . For re-ChIP assay, the DNA complexes were immunoprecipitated and eluted in the first-step ChIP using anti-YAP  antibody, followed by addition into 10mM DTT and incubation for 30 min at 37 °C. After centrifugation, the supernatant was diluted and immunoprecipitated using anti-TEAD1 antibody . The resulting precipitated DNA samples were subjected for qPCR analysis.The biotinylated probe targeting circ-YAP promoter was designed and synthesized, followed by incubation with the sonicated nuclear extracts at 4 °C overnight with agitation. The Streptavidin Magnetic Beads (Invitrogen) was added and incubated for 1 h at 4 °C. The beads were washed three times and the enriched proteins were eluted by 1×loading buffer, followed by western blot analysis of YAP and TEAD1 levels.Data are shown as mean ± standard deviation (SD). Two-tailed Student’s t-test was used to compare the results for any two preselected groups accounting for variance. The ROC curve was used to estimate the predictive accuracy. The survival curve of CRC patients was generated using Kaplan-Meier plotter, which was analyzed by Log-rank test. All statistical charts were generated by Graph Prism 7 software . A probability value less than 0.05 was considered statistically significant.To identify the key circRNAs with translational activity in CRC liver metastasis, we analyzed circRNA microarray and ribosome nascent-chain complex-bound RNA sequencing data. The overlapped results showed that circ-YAP was upregulated in CRC liver metastasis and might have translation potential Figure A, B. MorTo investigate the biological functions of circ-YAP, we used CRISPR/Cas13d technology to silence circ-YAP Figure A. As sho6A modification, we first evaluated the translational activity of IRES on circ-YAP. Two putative IRES sequences were inserted into dual luciferase vector system , affecting RNA stability, precursor splicing, polyadenylation, transport and translation initiation [6A motif “GGACA” proximal to the translation initiation site of circ-YAP, its mutation resulted in almost undetectable protein produced by circ-YAP, indicating that a single m6A is sufficient to drive circRNA translation, which is consistent with the previous evidence [6A-mutant one, promoted CRC liver metastasis, implying that circ-YAP functions in CRC depending on its translation potential. Interestingly, the expression trend of circ-YAP and YAP-220aa was not consistent in 7.5% CRC cases, this may be caused by the dysregulation of some m6A enzymes, or by the abnormal modification of the translation initiation complexs, etc. Additionally, although cytoplasmic localization is a prerequisite for RNA translation, 20% of circ-YAP was found in the nucleus, suggesting that circ-YAP has other roles besides coding proteins, and further investigations are needed.CircRNA regulates a wide range of biological processes in a context-dependent manner . The subcterized . Once thcterized , 32. m6Aitiation . Herein,evidence . MoreoveThe Hippo-YAP pathway is initially considered as an evolutionarily and functionally conserved modulator of organ size and growth with key roles in cell proliferation and differentiation . RecentlThere are several limitations in this work, with the major drawback being that the in vitro and in vivo studies were performed using human 2D cell line lines and do not truly reflect clinical tumor heterogeneity, cloning, growth and progression, the use of state-of-the-art 3D organoid systems will be helpful. In addition, our study included only retrospectively collected samples and information, the retrospective nature of the collection is associated with potential bias from variable treatments.Taken together, our study uncovers a hitherto unrecognized coupling between circ-RNA-encoded protein and Hippo/YAP signaling, with implications for the treatment of CRC liver metastasis.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3