Epitranscriptomic and expression profiling in Riccia fluitans across diverse environmental conditions

Impaired gene regulation lies at the heart of many disorders, including cancer. Gene expression is controlled through multiple mechanisms that are coordinated to ensure the proper and timely expression of each gene. From transcription to translation, every step of mRNA processing is controlled by interaction between the mRNA transcript and regulatory factors, such as RNA binding proteins or small noncoding RNAs. We now understand that these regulatory interactions can be modulated by RNA modifications. Presence of a modified nucleotide can create, or abrogate, sites for regulatory interactions by at least two distinct mechanisms: by inducing changes in RNA structure or through direct recognition by the RNA binding protein of the modified nucleotide. The RNA modification N6-methyladenosine (m6A) is present in several classes of RNAs, including mRNAs and lncRNAs, where it is the most abundant internal RNA modification. In mRNAs and lncRNAs, the m6A modification is present in a well-defined RNA motif, RRACH (R = A or G, H = A, C or U), and is enriched at specific transcript landmarks such as the last transcribed exon, near the STOP codon. The METTL3/METTL14 heterodimer is at the core of the large protein complex that catalyzes addition of m6A to mRNAs. To date, two enzymes capable of removing m6A have been identified: fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). Our studies on the role of m6A in ESCs revealed that thousands of messenger and long noncoding RNAs are m6A-modified, including transcripts encoding core pluripotency transcription factors such as Nanog, Sox2, and Myc. Our observations suggest that m6A marks unstable transcripts, including transcripts that need to be turned over upon differentiation. Loss of Mettl3 did not affect self-renewal, but resulted in impaired ESC exit from self-renewal towards differentiation to several lineages. Mettl3 KO cells did not differentiate into cardiomyocites or neurons in vitro, and subcutaneous injection of Mettl3 KO cells formed tumors consistent in morphology with teratomas that tended to be larger than tumors derived from wild-type cells. Furthermore, teratomas derived from Mettl3 KO cells were predominantly composed of poorly differentiated cells with very high mitotic indices and numerous apoptotic bodies, whereas wild-type cells differentiated predominantly into neuroectoderm. The role of m6A in facilitating transition between cell states is not limited to embryonic stem cells. The m6A interacting protein YTHDC2 is required for a clean transition from mitosis to meiosis in male and female germ cells. Ythdc2-/- male germ cells attempt to enter meiotic prophase but appear to have a mixed identity, maintaining expression of mitotic Cyclin A2 and failing to properly express many meiotic markers. Instead of executing meiotic prophase, the cells attempt an abnormal mitotic-like division, then quickly die. YTHDC2 is required during the early stages of meiotic entry in mice to both downregulate the previous mitotic program and facilitate proper expression of meiotic and differentiation genes. We propose that m6A makes the transition between cell states possible by facilitating a reset mechanism between stages, as occurs in ESCs and during gametogenesis. In contrast to epigenetic mechanisms that provide cellular memory of gene expression states, m6A enforces the transience of genetic information, helping cells to forget the past and thereby embrace the future. Citation Format: Pedro J. Batista. RNA modifications and cell identity: The importance of forgetting the past to embrace the future [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY43-02.

RNA m6A Modification in Cancers: Molecular Mechanisms and Potential Clinical Applications

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers complex host responses, including alterations in RNA transcription and modification. Understanding these changes is crucial for elucidating viral pathogenesis and identifying potential therapeutic targets. We used direct RNA sequencing to comprehensively profile the transcriptomic and epitranscriptomic landscapes of human HEK-AT cells infected with SARS-CoV-2 at 8 h post-infection, compared to mock controls. We analysed viral and host transcriptomes, focusing on gene and transcript expression, isoform usage and RNA m6A modifications. Viral RNA sequencing reads showed 3′ end-biassed coverage indicative of subgenomic RNA synthesis, with high expression of N gene subgenomic RNA reads. Sixteen m6A modification sites were consistently identified in the viral genome, primarily within the ORF1ab and S genes. In the human transcriptome, we found 254 positions with significantly altered m6A modification rates, with 119 showing decreased modification and 135 showing increased modification in infected cells. Genes with decreased m6A modifications were enriched in the neurotrophin signalling pathway. Transcript-level analysis identified 19 upregulated and 12 downregulated transcripts. Notably, transcript discovery and quantification revealed a novel isoform of the HIST1H2BK gene, which was significantly more expressed in infected cells compared to mock controls. Isoform switching analysis revealed 24 significant switches involving 21 genes, implicating mitochondrial reprogramming and immune-related pathways. In conclusion, this study provides a detailed, direct RNA sequencing-based characterization of host–virus RNA interactions, revealing key insights into SARS-CoV-2 infection mechanisms and potential therapeutic targets.

Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergo in response to land-water transition. A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant’s resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions. These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution.These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant’s dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

The growth and development of plants are the result of the interplay between the internal developmental programming and plant-environment interactions. Gene expression regulations in plants are made up of multi-level networks. In the past few years, many studies were carried out on co- and post-transcriptional RNA modifications, which, together with the RNA community, are collectively known as the "epitranscriptome." The epitranscriptomic machineries were identified and their functional impacts characterized in a broad range of physiological processes in diverse plant species. There is mounting evidence to suggest that the epitranscriptome provides an additional layer in the gene regulatory network for plant development and stress responses. In the present review, we summarized the epitranscriptomic modifications found so far in plants, including chemical modifications, RNA editing, and transcript isoforms. The various approaches to RNA modification detection were described, with special emphasis on the recent development and application potential of third-generation sequencing. The roles of epitranscriptomic changes in gene regulation during plant-environment interactions were discussed in case studies. This review aims to highlight the importance of epitranscriptomics in the study of gene regulatory networks in plants and to encourage multi-omics investigations using the recent technical advancements.

Chapter 1 - Epigenetic processes and DNA repair in embryonic stem cells

The N6-methyladenine (m6A) modification is the most common modification of messenger RNAs in eukaryotes and has crucial roles in multiple cancers, including bladder cancer (BLCA). This paper aimed to probe the molecular mechanism of zinc-finger CCCH-type containing 13 (ZC3H13)-mediated N6-methyladenine (m6A) modification in BLCA progression via autophagy. Differential expression of ZC3H13 in BLCA was analyzed by the bioinformatics database. ZC3H13 expression in BLCA tissues and cell lines was determined, and malignant behaviors of BLCA cells were examined in vitro and in vivo. ZC3H13 was decreased in BLCA tissues and cell lines relative to adjacent tissues and normal uroepithelial cells. ZC3H13 overexpression restricted BLCA cell growth in vitro and curbed BLCA development in vivo. ZC3H13 promoted the mRNA stability of paraja ring finger 2 (PJA2) through m6A modification, leading to the ubiquitination degradation of the kinase suppressor of Ras 1 (KSR1). Knockdown of PJA2 and overexpression of KSR1 reversed the inhibitory effect of ZC3H13 on BLCA progression. ZC3H13 degraded KSR1 through m6A modification of PJA2, promoted cell autophagy, and repressed BLCA progression. Overall, ZC3H13 promotes the mRNA stability of PJA2 through m6A modification to degrade KSR1, thereby promoting autophagy in BLCA.

<p>Soil fauna are now recognized as key components of soil and plant health, but how soil fauna modulates or even dictates plant stress responses remains unknown.Prior studies found the impacts of soil fauna on the plantprioritize either growth or defence depends on the feeding types of herbivores, suggesting that aboveground responses to herbivores and soil fauna are interconnected.However, the molecular and metabolism mechanism underlying this relationship is unclear.Theoretically, soil biotic interactions modified by soil fauna can drive above-ground responses, but there is still shortage of experimental evidence. Here, we aimed to test whether and how the plant survival strategies subjective to different feeding types of herbivores, were affected by rhizosphere microbial interactions exerted by soil fauna. We hypothesized that aboveground stress responses in plants can be orchestrated through coordinating belowground biotic interaction by soil fauna-root commensals.</p><p>First, we set out a complete factor design experiment under field condition by manipulating earthworms (<em>Metaphire guillelmi</em>) and the types of above-ground insects (cell-feeding, thrips: <em>Frankliniella occidentalis</em>; phloem-sucking, aphid: <em>Myzus persicae</em>). Soil physicochemical analysis and leaf RNA-seq were used to expound how earthworms dictates the trade-off between plant growth and defence. Second, to further explore the soil biotic-mediate pathway in the presence of earthworms. A microcosm experiment was performed by using a re-inoculated different soil community (obtained from 1000 and 20 μm sieves, respectively) collected from the field, with and without earthworms in the absence of herbivores. We used amplicon sequencing and plant metabolite analysis to elucidate how earthworm function by shaping rhizosphere biotic interactions.</p><p>We report here that belowground commensals can orchestrate aboveground stress responses in plants through biotic interaction. Earthworms suppressed thrips number, consistent with a resource-cost model. Contrary, earthworms promoted aphids’ number through the hormone antagonism model. Notably, soil biotic properties affected plant performance rather than soil abiotic properties mediated by earthworm. The microcosm experiment verified that rhizosphere microbial diversity and community structure shaped by earthworms reformed plant performance. In inoculated soil conditioned by earthworms, soil microbiome tended to be bacterial community towards primary metabolic processes, strengthening the interaction between fungi and bacteria in the rhizosphere. Further, the strengthened rhizosphere microbial interactions were increased plant shoot biomass and soluble sugar accumulation but decreased JA content. Taken together, effects of soil fauna on plant growth-defence relationships depend on the feeding types of herbivores, mainly attributing to the shifts in rhizosphere microbial interactions. Phenotypic plasticity and aboveground stress responses in plants can therefore be governed by soil fauna-root commensals.</p>

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m5C), in the transcriptome remains unclear. Aberrant regulation of 5-methylcytosine turnover in cancer may affect posttranscriptional modifications in coding and noncoding RNAs in disease pathogenesis. Mutations in NSUN2 have been reported as drivers of neurodevelopmental disorders in mice, and upregulated expression of NSUN2 in tumors of the breast, bladder, and pancreas has been reported. In this study, we conducted mRNA whole transcriptomic bisulfite sequencing to categorize NSUN2 target sites in the mRNA of human pancreatic cancer cells. We identified a total of 2829 frequent m5C sites in mRNA from pancreatic cancer cells. A total of 90.9% (2572/2829) of these m5C sites were mapped to annotated genes in autosomes and sex chromosomes X and Y. Immunohistochemistry staining confirmed that the NSUN2 expression was significantly upregulated in cancer lesions in the LSL-KrasG12D/+;Trp53fl/fl;Pdx1-Cre (KPC) spontaneous pancreatic cancer mouse model induced by Pdx1-driven Cre/lox system expressing mutant KrasG12D and p53 deletion. The in vitro phenotypic analysis of NSUN2 knockdown showed mild effects on pancreatic cancer cell 2D/3D growth, morphology and gemcitabine sensitivity in the early phase of tumorigenesis, but cumulative changes after multiple cell doubling passages over time were required for these mutations to accumulate. Syngeneic transplantation of NSUN2-knockdown KPC cells via subcutaneous injection showed decreased stromal fibrosis and restored differentiation of ductal epithelium in vivo. SIGNIFICANCE: Transcriptome-wide mRNA bisulfite sequencing identified candidate m5C sites of mRNAs in human pancreatic cancer cells. NSUN2-mediated m5C mRNA metabolism was observed in a mouse model of pancreatic cancer. NSUN2 regulates cancer progression and epithelial differentiation via mRNA methylation.

N6-methyl-adenosine (m6a) is involved in the occurrence and development of various diseases such as autogenic immune disease and tumors. Methyltransferases regulate primary (pri)-microRNA (miRNA/miR) processing by mediating m6a modifications, consequently affecting pathological processes including immune-related diseases by regulating both innate and adaptive immune cells. However, the roles of m6a on the biological functions of bone marrow mesenchymal stem cells (BMSCs) remain to be elucidated. The relative expression levels of methyltransferase-like 14 (METTL14) and other methyltransferases, demethylases, and miR-873 in bone samples from patients with osteoporosis and from normal individuals were measured by reverse transcription-quantitative PCR. Cell Counting Kit-8 assay was used to examine the proliferation of BMSCs. Co-immunoprecipitation (Co-IP) was used to investigate the binding of METTL14 to DiGeorge syndrome critical region 8 (DGCR8). RNA immunoprecipitation (RIP) was used to examine the binding of METTL14 to pri-miR-873. METTL14 and m6a modifications were highly detected in patients with osteoporosis compared with the controls. Co-IP results indicated that silencing of METTL14 reduced METTL14 and m6a modification levels in BMSCs. Downregulation of METTL14 significantly promoted the proliferation of BMSCs. RIP results suggested that METTL14/m6a methylation modification promoted the processing of pri-miR-873 by binding to DGCR8 in BMSCs. Furthermore, overexpression of miR-873 inhibited the proliferation of BMSCs. The results also showed that miR-873 mimics significantly inhibited the proliferation in small interfering (si)-METTL14 transfected BMSCs; however, miR-873 inhibitors markedly promoted the proliferation of si-METTL14 transfected BMSCs. METTL14 and m6a modifications were upregulated in osteoporosis samples. METTL14 promoted the processing of pri-miR-873 into mature miR-873 by regulating m6a modification. Furthermore, overexpression of miR-873 significantly inhibited the proliferation of BMSCs. Therefore, the METTL14/m6a/miR-873 axis may be a potential target for the treatment of osteoporosis.

SARS-CoV-2 infection triggers complex host responses, including alterations in RNA transcription, and modification. Understanding these changes is crucial for elucidating viral pathogenesis and identifying potential therapeutic targets. We used direct RNA sequencing to comprehensively profile the transcriptomic and epitranscriptomic landscapes of human HEK-AT cells infected with SARS-CoV-2 at 8 hours post-infection, compared to mock controls. We analyzed viral and host transcriptomes, focusing on gene and transcript expression, isoform usage, and RNA m6A modifications. Viral RNA sequencing reads showed 3’ end-biased coverage indicative of subgenomic RNA synthesis, with high expression of N gene subgenomic RNA reads. Sixteen m6A modification sites were consistently identified in the viral genome, primarily within the ORF1ab and S genes. In the human transcriptome, we found 254 positions with significantly altered m6A modification rates, with 119 showing decreased modification and 135 showing increased modification in infected cells. Genes with decreased m6A modifications were enriched in the neurotrophin signaling pathway. Transcript-level analysis identified 19 upregulated and 12 downregulated transcripts. Notably, transcript discovery and quantification revealed a novel isoform of the HIST1H2BK gene, which was significantly more expressed in infected cells compared to mock controls. Isoform switching analysis revealed 24 significant switches involving 21 genes, implicating mitochondrial reprogramming and immune-related pathways. In conclusion, this study provides a detailed, direct RNA sequencing-based characterization of host-virus RNA interactions, revealing key insights into SARS-CoV-2 infection mechanisms and potential therapeutic targets.

Multiple roles of p53 in cancer development: Regulation of tumor microenvironment, m6A modification and diverse cell death mechanisms

With advanced technologies to map RNA modifications, our understanding of them has been revolutionized, and they are seen to be far more widespread and important than previously thought. Current next-generation sequencing (NGS)-based modification profiling methods are blind to RNA modifications and thus require selective chemical treatment or antibody immunoprecipitation methods for particular modification types. They also face the problem of short read length, isoform ambiguities, biases and artifacts. Direct RNA sequencing (DRS) technologies, commercialized by Oxford Nanopore Technologies (ONT), enable the direct interrogation of any given modification present in individual transcripts and promise to address the limitations of previous NGS-based methods. Here, we present the first ONT-based database of quantitative RNA modification profiles, DirectRMDB, which includes 16 types of modification and a total of 904,712 modification sites in 25 species identified from 39 independent studies. In addition to standard functions adopted by existing databases, such as gene annotations and post-transcriptional association analysis, we provide a fresh view of RNA modifications, which enables exploration of the epitranscriptome in an isoform-specific manner. The DirectRMDB database is freely available at: http://www.rnamd.org/directRMDB/.

BackgroundRiccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition.ResultsA significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant’s resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions.ConclusionsThese insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant’s dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N6-methyladenosine (m6A), an epigenetic modification prevalent in mRNAs, is implicated in the transcriptional regulation during spermatogenesis. However, the dynamics of m6A modification in non-rodent mammalian species remains unclear. Here, we systematically investigated the profile and role of m6A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m6A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m6A pattern between porcine and murine genes with spermatogenic function. We found that m6A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m6A modification. Our data show that m6A plays the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point are three genes (SETDB1, FOXO1, and FOXO3) that are crucial to the determination of the fate of SSCs. To the best of our knowledge, this study for the first time uncovers the expression profile and role of m6A during spermatogenesis in large animals and provides insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.