A versatile tRNA modification-sensitive northern blot method with enhanced performance

The ~22 mitochondrial and ~45 cytosolic tRNAs contain several dozen different posttranscriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression and their deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our lab developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ~10 years ago which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of an DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32 and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.

The Novel Role of Arsenic (+3 oxidation state) Methyltransferase in Arsenic Genotoxicity

Background: Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in methylation metabolism of arsenic. It is closely related to DNA methylation, but little is known about the novel molecular mechanisms.Methods: 79 workers and 41 individuals in the control group were recruited. Arsenic, relative indexes, 28 relative RNAs, and base modifications of exon 5-8 of p53 were detected. Enzyme linked immunosorbent assay(ELISA) was performed to detect the expression of AS3MT protein in all subjects. A series of methods were used to analyze the relationships between them. The AS3MT protein was detected in A549 and 16HBE cells after treated using sodium arsenite, MMA and DMA for 48 hours. Small interfering RNA (siRNA) transfection was used to investigate the role of AS3MT in arsenite-induced tumorigenesis. The cell proliferation and apoptosis were assessed with MTT assay, EdU assay, HO/PI double staining and JC-1 assay. The real-time quantitative PCR (qRT-PCR) and Western Blot analyses were used to evaluate the expression of genes. The p53 luciferase reporter gene assay and Co-immunoprecipitation (Co-IP) were used to identify the interactions of target proteins.Results: AS3MT RNA is closely related to p53, a series of ncRNAs and mRNAs, and likely to have causal correlations. Base modifications of p53, miR-548 and miR-190 have significant distinctive effects, but arsenic may play limited roles. AS3MT is over expression in lung cancer patients who have not exposed to arsenic, human lung adenocarcinoma and bronchial epithelial cells with arsenic treatment for 48h. AS3MT protein is induced in arsenic exposed population. Down regulation of AS3MT inhibit proliferation and promotes apoptosis of cells. Mechanistically, AS3MT specifically bind with c-Fos, and block the binding ability between c-Fos and c-Jun. Additionally, knockdown of AS3MT mediated by siRNA enhance the phosphorylation level of p53 Ser392 through activating p38 MAPK. These probably lead to activation of p53 signaling and up regulation in downstream targets, such as p21, Fas, Puma and Bax.Discussion: Here showed that AS3MT RNA plays a great role in the genotoxicity and carcinogenesis which started by arsenic, but influenced by other factors. Up regulation of AS3MT can directly act on cell, and affect cell proliferation and apoptosis through activation of p53 signaling and up regulation in downstream targets.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

A DNA-repair pathway can affect transcriptional noise to promote cell fate transitions

Stochastic fluctuations in gene expression (‘noise’) are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base-excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA-repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean-expression levels. This amplified expression noise originates from shorter duration, higher intensity, transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we term Discordant Transcription through Repair (DiThR; pronounced /’dither’/), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. Yet, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications poorly understood. We developed a 13C labeling approach, 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleosides, and showed the distinct kinetics of N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits a distinct turnover in small RNAs and free ribonucleosides when compared to the known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of origin of modified RNAs at steady-state and their dynamics under non-stationary conditions.

Nanopore callers for epigenetics from limited supervised data

Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5mC and 6mA. These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA k-mer backgrounds—a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with Hidden Markov Models (HMMs) that cannot make successful calls for k-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen k-mer contexts. Indeed, herein we demonstrate that a common DNN approach (DeepSignal) outperforms a common HMM approach (Nanopolish) in the incomplete data setting. Furthermore, we propose a novel hybrid HMM-DNN approach, Amortized-HMM, that outperforms both the pure HMM and DNN approaches on 5mC calling when the training data are incomplete. Such an approach is expected to be useful for calling 5hmC and combinations of cytosine modifications, where complete training data are not likely to be available.

Cancer Biomarkers Discovery of Methylation Modification With Direct High-Throughput Nanopore Sequencing

Cancer is a complex disease, driven by a combination of genetic and epigenetic alterations. DNA and RNA methylation modifications are the most common epigenetic events that play critical roles in cancer development and progression. Bisulfite converted sequencing is a widely used technique to detect base modifications in DNA methylation, but its main drawbacks lie in DNA degradation, lack of specificity, or short reads with low sequence diversity. The nanopore sequencing technology can directly detect base modifications in native DNA as well as RNA without harsh chemical treatment, compared to bisulfite sequencing. Furthermore, CRISPR/Cas9-targeted enrichment nanopore sequencing techniques are straightforward and cost-effective when targeting genomic regions are of interest. In this review, we mainly focus on DNA and RNA methylation modification detection in cancer with the current nanopore sequencing approaches. We also present the respective strengths, weaknesses of nanopore sequencing techniques, and their future translational applications in identification of epigenetic biomarkers for cancer detection and prognosis.

DNA METHYLATION PROFILING OF A CNIDARIAN-ALGAL SYMBIOSIS USING NANOPORE SEQUENCING

Symbiosis with protists is common among cnidarians such as corals and sea anemones, and is associated with homeostatic and phenotypic changes in the host that could have epigenetic underpinnings, such as methylation of CpG dinucleotides. We leveraged the sensitivity to base modifications of nanopore sequencing to probe the effect of symbiosis with the chlorophyte Elliptochloris marina on methylation in the sea anemone Anthopleura elegantissima. We first validated the approach by comparison of nanopore-derived methylation levels with CpG depletion analysis of a published transcriptome, finding that high methylation levels are associated with CpG depletion as expected. Next, using reads generated exclusively from aposymbiotic anemones, a largely complete draft genome comprising 243 Mb was assembled. Reads from aposymbiotic and symbiotic sea anemones were then mapped to this genome and assessed for methylation using the program Nanopolishwhich detects signal disruptions from base modifications as they pass through the nanopore. Based on assessment of 452,841 CpGs for which there was adequate read coverage (approximately 8% of the CpGs in the genome), symbiosis with E. marina was, surprisingly, associated with only subtle changes in the host methylome. However, we did identify one extended genomic region with consistently higher methylation among symbiotic individuals. The region was associated with a DNA polymerase zeta that is noted for its role in translesion synthesis, which opens interesting questions about the biology of this symbiosis. Our study highlights the power and relative simplicity of nanopore sequencing for studies of nucleic acid base modifications in non-model species.

Deciphering Epitranscriptome: Modification of mRNA Bases Provides a New Perspective for Post-transcriptional Regulation of Gene Expression

Gene regulation depends on dynamic and reversibly modifiable biological and chemical information in the epigenome/epitranscriptome. Accumulating evidence suggests that messenger RNAs (mRNAs) are generated in flashing bursts in the cells in a precisely regulated manner. However, the different aspects of the underlying mechanisms are not fully understood. Cellular RNAs are post-transcriptionally modified at the base level, which alters the metabolism of mRNA. The current understanding of epitranscriptome in the animal system is far ahead of that in plants. The accumulating evidence indicates that the epitranscriptomic changes play vital roles in developmental processes and stress responses. Besides being non-genetically encoded, they can be of reversible nature and involved in fine-tuning the expression of gene. However, different aspects of base modifications in mRNAs are far from adequate to assign the molecular basis/functions to the epitranscriptomic changes. Advances in the chemogenetic RNA-labeling and high-throughput next-generation sequencing techniques are enabling functional analysis of the epitranscriptomic modifications to reveal their roles in mRNA biology. Mapping of the common mRNA modifications, including N6-methyladenosine (m6A), and 5-methylcytidine (m5C), have enabled the identification of other types of modifications, such as N1-methyladenosine. Methylation of bases in a transcript dynamically regulates the processing, cellular export, translation, and stability of the mRNA; thereby influence the important biological and physiological processes. Here, we summarize the findings in the field of mRNA base modifications with special emphasis on m6A, m5C, and their roles in growth, development, and stress tolerance, which provide a new perspective for the regulation of gene expression through post-transcriptional modification. This review also addresses some of the scientific and technical issues in epitranscriptomic study, put forward the viewpoints to resolve the issues, and discusses the future perspectives of the research in this area.