RNA Bisulfite Sequencing Research Articles

Splice_sim: a nucleotide conversion-enabled RNA-seq simulation and evaluation framework

Nucleotide conversion RNA sequencing techniques interrogate chemical RNA modifications in cellular transcripts, resulting in mismatch-containing reads. Biases in mapping the resulting reads to reference genomes remain poorly understood. We present splice_sim, a splice-aware RNA-seq simulation and evaluation pipeline that introduces user-defined nucleotide conversions at set frequencies, creates mixture models of converted and unconverted reads, and calculates mapping accuracies per genomic annotation. By simulating nucleotide conversion RNA-seq datasets under realistic experimental conditions, including metabolic RNA labeling and RNA bisulfite sequencing, we measure mapping accuracies of state-of-the-art spliced-read mappers for mouse and human transcripts and derive strategies to prevent biases in the data interpretation.

NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization.

NOP2/Sun domain 2 (NSUN2) is one of the important RNA methyltransferases catalyzing 5-methylcytosine (m5C) formation and participates in many critical bioprocesses. However, the roles and underlying molecular mechanisms of NSUN2-mediated m5C modification in colorectal cancer (CRC) remain unclear. To explore the NSUN2 expression in CRC, fresh tissue samples were collected and Nsun2 knockout mouse was constructed. In vitro and in vivo functional assays were conducted to assess the role of NSUN2. RNA array and bisulfite sequencing were used to investigate the potential targets. The mechanisms of NSUN2 function on SKIL were identified by m5C-methylated-RNA immunoprecipitation and RNA stability assays. Additionally, tissue microarray analysis was conducted and patient-derived tumour xenograft mouse (PDX) models were used to define the potential therapeutic targets. NSUN2 was highly expressed in CRC and correlated with poor CRC patient survival. Moreover, silencing NSUN2 suppressed CRC tumourigenesis and progression in Nsun2 knockout mouse models. In vitro and in vivo studies suggested that NSUN2 promoted colorectal cancer cell growth. Mechanistically, SKI-like proto-oncogene (SKIL) is positively regulated by NSUN2, and the NSUN2-SKIL axis is clinically relevant to CRC. NSUN2 induced m5C modification of SKIL and stabilized its mRNA, which was mediated by Y-box binding protein 1 (YBX1). Elevated SKIL levels increased transcriptional coactivator with PDZ-binding motif (TAZ) activation. Our findings highlight the importance of NSUN2 in the initiation and progression of CRC via m5C-YBX1-dependent stabilization of the SKIL transcript, providing a promising targeted therapeutic strategy for CRC.

Epigenetically upregulated NSUN2 confers ferroptosis resistance in endometrial cancer via m5C modification of SLC7A11 mRNA

Endometrial cancer (EC) is a prevalent gynecological malignancy worldwide, and 5-methylcytosine (m5C) modification of mRNA is a crucial epigenetic modification associated with the development and occurrence of several cancers. However, the precise function of m5C modification in EC remains elusive. This study aimed to investigate the expression and clinical significance of the primary m5C modification writer, NSUN2, in EC. Our findings indicated that NSUN2 exhibited a substantial up-regulation in EC as a result of an epigenetic augmentation in H3K4me3 levels within the promoter region, which was triggered by the down-regulation of KDM5A. Moreover, gain- and loss-of-function experiments revealed the role of NSUN2 in enhancing m5C modification of mRNA, thereby promoting EC cell proliferation. RNA bisulfite sequencing and transcriptomic sequencing were employed to elucidate the involvement of NSUN2 in the regulation of ferroptosis. Subsequent in vitro experiments confirmed that the knockdown of NSUN2 significantly up-regulated the levels of lipid peroxides and lipid ROS in EC cells, thereby augmenting the susceptibility of EC to ferroptosis. Mechanistically, NSUN2 stimulated the m5C modification of SLC7A11 mRNA, and the m5C reader YBX1 exhibited direct recognition and binding to the m5C sites on SLC7A11 mRNA via its internal cold shock domain (CSD), leading to an increase in SLC7A11 mRNA stability and elevated levels of SLC7A11. Additionally, rescue experiments showed that NSUN2 functioned as a suppressor of ferroptosis, which was dependent on SLC7A11. Overall, targeting the NSUN2/SLC7A11 axis inhibited tumor growth by increasing lipid peroxidation and ferroptosis of EC cells both in vitro and in vivo. Therefore, our study provides new insight into the role of NSUN2, suggesting that NSUN2 may serve as a prognostic biomarker and therapeutic target in patients with EC.

Global DNA Methylation, miRNA, and mRNA Profiles in Sheep Skeletal Muscle Promoted by Hybridization.

With the development of high-throughput sequencing technology, several nongenetic variations, including noncoding RNAs such as miRNAs, and DNA methylation, have been found to play an important role in animal muscle development and fat metabolism. In this study, Southdown and Suffolk were selected as male parents for hybridization with Hu sheep (Southdown × Hu (NH), Suffolk × Hu (SH), and Hu × Hu (HH)). RNA sequencing, bisulfite sequencing, and small-RNA sequencing were used to study the methylation patterns and differences in miRNA and mRNA expression in the F1 sheep longissimus dorsi muscle tissue. We identified 765 differentially expressed genes (DEGs), 10,161 differentially methylated regions (DMRs), and 164 differentially expressed miRNAs, which were significantly enriched in AMPK signaling, fatty acid degradation, metabolism, and other related pathways (P < 0.05). In addition, we constructed a DNA methylation-mRNA and miRNA-mRNA coexpression network. A total of 42 common genes were identified from DMRs and DEGs. Importantly, we predicted that 33 differentially expressed miRNAs directly or indirectly targeted the SLC27A6. The data obtained in this study provide useful information and evidence to support further understanding of the miRNA and DNA methylation of key genes regulating muscle growth and fat metabolism in hybrid sheep populations.

Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer

BackgroundRNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors.MethodsCombining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity.ResultsIn this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors.ConclusionThe 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability.

Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer

BackgroundRNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear.MethodsThe correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays.ResultsRNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1).ConclusionsOur study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

Positive epigenetic regulation loop between AR and NSUN2 promotes prostate cancer progression.

BackgroundProstate cancer (PCa) is a major type of cancer in man worldwide. Androgen deprivation therapy (ADT) and the next‐generation androgen receptor (AR) pathway inhibitors have acquired great success in treating PCa. However, patients treated with ADT or AR targeted therapy are inevitably developing into castration‐resistant prostate cancer (CRPC) or becoming drug resistance. The role of mRNA 5‐methylcytosine (m5C) modification in cancers is largely unknown. This study aimed to explore the role of the m5C methyltransferase NSUN2 in Prostate cancer (PCa).MethodsThe expression of NSUN2 and its clinicopathological impact were evaluated in PCa cohorts. The effect of NSUN2 on the biological characteristics of PCa cells was investigated on the basis of gain‐offunction and loss‐of‐function analyses. Subcutaneous models further uncovered the role of NSUN2 in tumor growth. Epi‐transcriptome assays with RNA bisulfite sequencing (RNA‐BisSeq) analysis and in vitro enzyme reaction assays were performed to validate the targeted effect of NSUN2 on AR. AR‐binding sites in the NSUN2 promoter were investigated by ChIP and luciferase assays to uncover the interplay between NSUN2 and AR signaling. RIP‐qPCR and EMSA methods were performed to confirm that YBX1 binds to AR m5C sites.ResultsNSUN2 is highly expressed in PCa and predicts poor outcome. NSUN2 plays roles as a PCa oncogene both in vitro and in vivo. Depletion of NSUN2 results in decreased expression and activities of AR, including AR‐V7. Mechanistically, NSUN2 posttranscriptionally stabilized AR by cluster m5C modification in a m5CYBX1‐dependent manner. Strikingly, treatment with enzalutamide, an effective AR inhibitor, reduces NSUN2 expression and decreases the m5C modification level in prostate cancer cells. Finally, we found that AR transcriptionally regulates NSUN2.ConclusionNSUN2 stabilizes AR mRNA through cluster 5‐methylcytosine modification and activates a positive feedback loop to promote prostate cancer.

Neuronal Depolarization Induced RNA m5C Methylation Changes in Mouse Cortical Neurons.

Simple SummaryWhen neurons are activated, two waves of gene expression changes occur to achieve neuronal adaptation to their environment. Currently, little is known about the changes in post-transcriptional modifications of neuronal transcripts in response to environmental stimuli. This study aims to investigate the dynamics of m5C modification on neuronal mRNAs upon depolarization using potassium chloride. We found that neuronal mRNAs carrying m5C modifications are enriched for genes with important neuronal functions. The numbers of m5C sites and methylated transcripts increased at 2 h after neuronal depolarization and slightly dropped at 6 h. Interestingly, for differentially methylated genes, a negative correlation between the levels of gene expression and RNA methylation is more prevalent. We anticipate our findings may shed new lights on post-transcriptional regulation in activated neurons, and the transcriptomic plus epi-transcriptomic datasets generated in this study would provide a valuable resource to the scientific community.Neuronal activity is accomplished via substantial changes in gene expression, which may be accompanied by post-transcriptional modifications including RNA cytosine-5 methylation (m5C). Despite several reports on the transcriptome profiling of activated neurons, the dynamics of neuronal mRNA m5C modification in response to environmental stimuli has not been explored. Here, we provide transcriptome-wide maps of m5C modification, together with gene expression profiles, for mouse cortical neurons at 0 h, 2 h, and 6 h upon membrane depolarization. Thousands of differentially expressed genes (DEGs) were identified during the neuronal depolarization process. In stimulated neurons, the majority of early response genes were found to serve as expression regulators of late response genes, which are involved in signaling pathways and diverse synaptic functions. With RNA bisulfite sequencing data, a union set of 439 m5C sites was identified with high confidence, and approximately 30% of them were shared by neurons at all three time points. Interestingly, over 41% of the m5C sites showed increased methylation upon neuronal activation and were enriched in transcripts coding for proteins with synaptic functions. In addition, a modest negative correlation was observed between RNA expression and methylation. In summary, our study provided dynamic transcriptome-wide landscapes of RNA m5C methylation in neurons, and revealed that mRNA m5C methylation is associated with the regulation of gene expression.

Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine Reprogramming During Early Human Development

After implantation, complex and highly specialized molecular events render functionally distinct organ formation, whereas how the epigenome shapes organ-specific development remains to be fully elucidated. Here, nano-hmC-Seal, RNA bisulfite sequencing (RNA-BisSeq), and RNA sequencing (RNA-Seq) were performed, and the first multilayer landscapes of DNA 5-hydroxymethylcytosine (5hmC) and RNA 5-methylcytosine (m5C) epigenomes were obtained in the heart, kidney, liver, and lung of the human foetuses at 13–28 weeks with 123 samples in total. We identified 70,091 and 503 organ- and stage-specific differentially hydroxymethylated regions (DhMRs) and m5C-modified mRNAs, respectively. The key transcription factors (TFs), T-box transcription factor 20 (TBX20), paired box 8 (PAX8), krueppel-like factor 1 (KLF1), transcription factor 21 (TCF21), and CCAAT enhancer binding protein beta (CEBPB), specifically contribute to the formation of distinct organs at different stages. Additionally, 5hmC-enriched Alu elements may participate in the regulation of expression of TF-targeted genes. Our integrated studies reveal a putative essential link between DNA modification and RNA methylation, and illustrate the epigenetic maps during human foetal organogenesis, which provide a foundation for an in-depth understanding of the epigenetic mechanisms underlying early development and birth defects.

RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.

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.

Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.

The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.

M5C-Atlas: a comprehensive database for decoding and annotating the 5-methylcytosine (m5C) epitranscriptome.

5-Methylcytosine (m5C) is one of the most prevalent covalent modifications on RNA. It is known to regulate a broad variety of RNA functions, including nuclear export, RNA stability and translation. Here, we present m5C-Atlas, a database for comprehensive collection and annotation of RNA 5-methylcytosine. The database contains 166 540 m5C sites in 13 species identified from 5 base-resolution epitranscriptome profiling technologies. Moreover, condition-specific methylation levels are quantified from 351 RNA bisulfite sequencing samples gathered from 22 different studies via an integrative pipeline. The database also presents several novel features, such as the evolutionary conservation of a m5C locus, its association with SNPs, and any relevance to RNA secondary structure. All m5C-atlas data are accessible through a user-friendly interface, in which the m5C epitranscriptomes can be freely explored, shared, and annotated with putative post-transcriptional mechanisms (e.g. RBP intermolecular interaction with RNA, microRNA interaction and splicing sites). Together, these resources offer unprecedented opportunities for exploring m5C epitranscriptomes. The m5C-Atlas database is freely accessible at https://www.xjtlu.edu.cn/biologicalsciences/m5c-atlas.

Genome-Wide DNA Methylation and Transcriptome Analyses Reveal Epigenetic and Genetic Mechanisms Underlying Sex Maintenance of Adult Chinese Alligator.

The sexes of Chinese alligators are determined during embryonic development and remain fixed thereafter. In this study, we investigated the genetic and epigenetic mechanisms underlying sex maintenance in Chinese alligators through RNA sequencing and bisulfite sequencing data analyses of the adult gonads. We identified the genes and pathways (e. g., DMRT1-SOX9-AMH pathway for males and oocyte meiotic maturation pathway for females) involved in male and female sex maintenance and gonadal development of adult Chinese alligators. In contrast to their expression patterns in the embryo, both DMRT1 and the steroid hormone biosynthesis related genes showed a male-biased expression in adult gonads. The overall DNA methylation density and level were higher in testes than in ovaries. Hypermethylation in the gene bodies enhanced the expression of male-biased genes (such as DMRT1-SOX9-AMH and steroid hormone biosynthesis related genes) in the testis, as opposed to the normalization of gene expression. Our results provide insights into the genetic and epigenetic mechanisms underlying sex maintenance in adult Chinese alligators, and are expected to contribute to the development of scientific programs for the successful conservation of this endangered species.

Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6.

The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3′UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.

YTHDF2 Binds to 5-Methylcytosine in RNA and Modulates the Maturation of Ribosomal RNA.

5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (m5C) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of m5C in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to m5C in RNA, albeit at a lower affinity than that toward N6-methyladenosine (m6A) in RNA, and this binding involves Trp432, a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for m6A recognition. RNA bisulfite sequencing results revealed that, after CRISPR-Cas9-mediated knockout of the YTHDF2 gene, the majority of m5C sites in rRNA (rRNA) exhibited substantially augmented levels of methylation. Moreover, we found that YTHDF2 is involved in pre-rRNA processing in cells. Together, our data expanded the functions of the YTHDF2 protein in post-transcriptional regulations of RNA and provided novel insights into the functions of m5C in RNA biology.

Advances in methods and software for RNA cytosine methylation analysis

Our understanding of RNA modifications has been growing rapidly over the last decade. Epitranscriptomics has recently emerged as an exciting, new field for understanding the fundamental mechanisms underlying RNA modifications and their impact on gene expression. Among the over one hundred different kinds of RNA modifications, cytosine methylation in mRNA (5-mrC) is now recognized as an important epigenetic mark that modulates mRNA transportation, translation, and stability at the post-transcriptional level. Across plant and animal species, recent studies have revealed the roles of mRNA cytosine methylation in several fundamental biological processes. In mammals, genome-wide profiling has determined thousands of mRNA transcripts carrying the 5-mrC modification in a tissue specific manner. Here, we summarize the experimental techniques that were exploited to determine 5-mrC in mRNA and the computational procedures implemented for RNA bisulfite sequencing data analysis.

Depletion of TRDMT1 affects 5-methylcytosine modification of mRNA and inhibits HEK293 cell proliferation and migration

Human TRDMT1 is a transfer RNA (tRNA) methyltransferase for cytosine-5 methylation and has been suggested to be involved in the regulation of numerous developmental processes. However, little is known about the molecular mechanisms or their biological significance. In this study, we investigated the effects of CRISPR-based TRDMT1 knockdown on phenotypes, mRNA m5C modifications and gene expression changes in HEK293 cells. We found that knockdown of TRDMT1 significantly inhibited cell proliferation and migration but had no effect on clonogenic potential. The inhibitory effects could be attenuated by re-expression of TRDMT1 in HEK293 cells. RNA sequencing (RNA-Seq) and RNA bisulfite sequencing (RNA-BisSeq) were performed in TRDMT1 knockdown and wild-type HEK293 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the differentially expressed genes were associated with the cell cycle, RNA transport, and RNA degradation and were enriched in cancer and Notch signaling pathways. We also found that TRDMT1 knockdown could change mRNA methylation levels. For the first time, these findings clarify the role of TRDMT1 in regulating mRNA methylation and inhibiting the proliferation and migration of HEK293 cells. These results provide new insights into a new function of TRDMT1 and elucidate the molecular mechanisms of aberrant RNA m5C during tumorigenesis.

Divulging the Enigma of Epitranscriptome in Plasmodium falciparum

In spite of prime posttranscriptional modifications, eukaryotic RNA is subjected to internal epitranscriptomic marks. Thus, these changes influence the rate of transcription which regulates the translational plasticity. However, RNA methylome and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, for the foremost a piece of our work demonstrated the cytosine methylation in the tRNA Asp at the C38 position of Plasmodium falciparum, which is accomplished by the tRNA aspartic acid methyl transferase1 (TRDMT1), a conserved homologue of DNA methyltransferase DNMT2. Through RNA bisulfite sequencing, we mapped the 5‐methylcytosines in endogenous tRNA Asp, followed by in‐vitro characterisation of TRDMT1. Accordingly, P. falciparum proteome divulged the significant role of polyaspartic acid repeat‐containing proteins and its abundance. Those proteins may play astonishing roles in host‐pathogen interactions. Further studies focus on the generation of TRDMT1 knock out parasites which may aid us to find out the critical role of TRDMT1 mediated C38 methylation of aspartic acid tRNA. In particular, translational regulation of essential proteins which could modulate the malaria parasite pathogenesis. On the other hand, the significant genes in P. falciparum show discrepancy in mRNA expression and its cognate protein, through which ‘time shift’ is displayed. Moreover, P. falciparum exhibits decelerated ring stage as a distinctive policy for drug resistance. In recent years, it is evidently proven that the epigenetic modifications are existed in eukaryotic mRNA, especially methylation at the nucleotide level and is the reason for time shift.In support, our preliminary data's on P. falciparum RNA implied the methylated nucleotides. We anticipate that parasites may modulate the translation process at the transcription level through methylation that could be installed by a multicomponent methyltransferase complex and follow‐up by the RNA binding proteins. Thus so, parasite might adopt the ‘just‐in‐time’ transcription and translation strategy for its emergence that are controlled by epigenetic machinery. Consequently, further mining of RNA methylome in parasite could be insightful about the host pathogen interaction. Hence, by cracking the epitrancriptome of P. falciparum, the underlying mechanisms of dynamic gene regulation of the parasite pathogenesis could be explored.Support or Funding InformationDepartment of science and technology, Government of India.Department of Biotechnology, Government of India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1-2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

5-Methylcytosine Analysis by RNA-BisSeq.

5-Methylcytosine (m5C) is a posttranscriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. Many known or novel m5C sites have been validated by using advanced high-throughput techniques combined with next-generation sequencing (NGS), especially RNA bisulfite sequencing (RNA-BisSeq). Here we introduce an optimized RNA-BisSeq method by using ACT random hexamers to prime the reverse transcription of bisulfite-treated RNA samples to detect the m5C sites.