Quantitative mapping of the mammalian epitranscriptome.

The epitranscriptome encompasses a diverse array of dynamic and reversible RNA modifications, affecting both coding and noncoding RNAs. Over 170 types of RNA chemical modifications have been identified, underscoring the need for innovative detection methods to deepen our understanding of RNA modification roles and mechanisms. In particular, the base resolution and quantitative information on RNA modifications are critical for understanding the regulation and functions of RNA modifications. Based on detection throughput and principles, existing quantitative RNA modification detection methods can be categorized into two groups, including next-generation sequencing and nanopore direct RNA sequencing. In this review, we focus on methodologies for elucidating the base resolution and stoichiometric information of RNA modifications. In addition, we further discuss the challenges and the potential prospects of the quantitative RNA modification detection methods.

Advances in the joint profiling technologies of 5mC and 5hmC.

DNA cytosine methylation, a crucial epigenetic modification, involves the dynamic interplay of 5-methylcytosine (5mC) and its oxidized form, 5-hydroxymethylcytosine (5hmC), generated by ten-eleven translocation (TET) DNA dioxygenases. This process is central to regulating gene expression, influencing critical biological processes such as development, disease progression, and aging. Recognizing the distinct functions of 5mC and 5hmC, researchers often employ restriction enzyme-based or chemical treatment methods for their simultaneous measurement from the same genomic sample. This enables a detailed understanding of the relationship between these modifications and their collective impact on cellular function. This review focuses on summarizing the technologies for detecting 5mC and 5hmC together but also discusses the limitations and potential future directions in this evolving field.

The Role of N6-methyladenosine Modification in Gametogenesis and Embryogenesis: Impact on Fertility.

The most common epigenetic modification of messenger ribonucleic acids (mRNAs) is N6-methyladenosine (m6A), which is mainly located near the 3' untranslated region of mRNAs, near the stop codons, and within internal exons. The biological effect of m6A is dynamically modified by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). By controlling post-transcriptional gene expression, m6A has a significant impact on numerous biological functions, including RNA transcription, translation, splicing, transport, and degradation. Hence, m6A influences various physiological and pathological processes, such as spermatogenesis, oogenesis, embryogenesis, placental function, and human reproductive system diseases. During gametogenesis and embryogenesis, genetic material undergoes significant changes, including epigenomic modifications such as m6A. From spermatogenesis and oogenesis to the formation of an oosperm and early embryogenesis, m6A changes occur at every step. m6A abnormalities can lead to gamete abnormalities, developmental delays, impaired fertilization, and maternal-to-zygotic transition blockage. Both mice and humans with abnormal m6A modifications exhibit impaired fertility. In this review, we discuss the dynamic biological effects of m6A and its regulators on gamete and embryonic development and review the possible mechanisms of infertility caused by m6A changes. We also discuss the drugs currently used to manipulate m6A and provide prospects for the prevention and treatment of infertility at the epigenetic level.

RNA base editors: The emerging approach of RNA therapeutics.

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m6A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.

Solanum aculeatissimum and Solanum torvum chloroplast genome sequences: a comparative analysis with other Solanum chloroplast genomes.

BACKGROUND: Solanum aculeatissimum and Solanum torvum belong to the Solanum species, and they are essential plants known for their high resistance to diseases and adverse conditions. They are frequently used as rootstocks for grafting and are often crossbred with other Solanum species to leverage their resistance traits. However, the phylogenetic relationship between S. aculeatissimum and S. torvum within the Solanum genus remains unclear. Therefore, this paper aims to sequence the complete chloroplast genomes of S. aculeatissimum and S. torvum and analyze them in comparison with 29 other previously published chloroplast genomes of Solanum species. RESULTS: We observed that the chloroplast genomes of S. aculeatissimum and S. torvum possess typical tetrameric structures, consisting of one Large Single Copy (LSC) region, two reverse-symmetric Inverted Repeats (IRs), and one Small Single Copy (SSC) region. The total length of these chloroplast genomes ranged from 154,942 to 156,004 bp, with minimal variation. The highest GC content was found in the IR region, while the lowest was in the SSC region. Regarding gene content, the total number of chloroplast genes and CDS genes remained relatively consistent, ranging from 128 to 134 and 83 to 91, respectively. Nevertheless, there was notable variability in the number of tRNA genes and rRNAs. Relative synonymous codon usage (RSCU) analysis revealed that both S. aculeatissimum and S. torvum preferred codons that utilized A and U bases. Analysis of the IR boundary regions indicated that contraction and expansion primarily occurred at the junction between SSC and IR regions. Nucleotide polymorphism analysis and structural variation analysis demonstrated that chloroplast variation in Solanum species mainly occurred in the LSC and SSC regions. Repeat sequence analysis revealed that A/T was the most frequent base pair in simple repeat sequences (SSR), while Palindromic and Forward repeats were more common in long sequence repeats (LSR), with Reverse and Complement repeats being less frequent. Phylogenetic analysis indicated that S. aculeatissimum and S. torvum belonged to the same meristem and were more closely related to Cultivated Eggplant. CONCLUSION: These findings enhance our comprehension of chloroplast genomes within the Solanum genus, offering valuable insights for plant classification, evolutionary studies, and potential molecular markers for species identification.

New directions for Ψ and m1A decoding in mRNA: deciphering the stoichiometry and function.

Over the past decade, advancements in epitranscriptomics have significantly enhanced our understanding of mRNA metabolism and its role in human development and diseases. This period has witnessed breakthroughs in sequencing technologies and the identification of key proteins involved in RNA modification processes. Alongside the well-studied m6A, Ψ and m1A have emerged as key epitranscriptomic markers. Initially identified through transcriptome-wide profiling, these modifications are now recognized for their broad impact on RNA metabolism and gene expression. In this Perspective, we focus on the detections and functions of Ψ and m1A modifications in mRNA and discuss previous discrepancies and future challenges. We summarize recent advances and highlight the latest sequencing technologies for stoichiometric detection and their mechanistic investigations for functional unveiling in mRNA as the new research directions.

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons.

Programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress premature termination codons (PTCs) that can lead to truncated and nonfunctional proteins. However, current methods to correct disease-associated PTCs suffer from low efficiency and limited precision. Here we develop RESTART v3, which uses near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold (range: 2.1- to 9.5-fold) higher editing efficiency than RESTART v2 in cultured cells and achieve functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense-to-nonsense codons, without affecting normal termination codons. Although off-target sites were detected, we did not observe changes to the coding information or the expression level of transcripts, and the overall natural tRNA abundance remained constant.

Programmable RNA base editing via targeted modifications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editors are powerful tools in biology and hold great promise for the treatment of human diseases. Advanced DNA base editing tools, such as cytosine base editor and adenine base editor, have been developed to correct permanent mistakes in genetic material. However, undesired off-target edits would also be permanent, which poses a considerable risk for therapeutics. Alternatively, base editing at the RNA level is capable of correcting disease-causing mutations but does not lead to lasting genotoxic effects. RNA base editors offer temporary and reversible therapies and have been catching on in recent years. Here, we summarize some emerging RNA editors based on A-to-inosine, C-to-U and U-to-pseudouridine changes. We review the programmable RNA-targeting systems as well as modification enzyme-based effector proteins and highlight recent technological breakthroughs. Finally, we compare editing tools, discuss limitations and opportunities, and provide insights for the future directions of RNA base editing.

Simultaneous single-cell analysis of 5mC and 5hmC with SIMPLE-seq.

Dynamic 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications to DNA regulate gene expression in a cell-type-specific manner and are associated with various biological processes, but the two modalities have not yet been measured simultaneously from the same genome at the single-cell level. Here we present SIMPLE-seq, a scalable, base resolution method for joint analysis of 5mC and 5hmC from thousands of single cells. Based on orthogonal labeling and recording of 'C-to-T' mutational signals from 5mC and 5hmC sites, SIMPLE-seq detects these two modifications from the same molecules in single cells and enables unbiased DNA methylation dynamics analysis of heterogeneous biological samples. We applied this method to mouse embryonic stem cells, human peripheral blood mononuclear cells and mouse brain to give joint epigenome maps at single-cell and single-molecule resolution. Integrated analysis of these two cytosine modifications reveals distinct epigenetic patterns associated with divergent regulatory programs in different cell types as well as cell states.

GLORI for absolute quantification of transcriptome-wide m6A at single-base resolution.

N6-methyladenosine (m6A) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative m6A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m6A intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 m6A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m6A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m6A biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.

Age-related noncanonical TRMT6-TRMT61A signaling impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity and differentiation bias toward myeloid lineages. However, the molecular mechanism behind HSC aging remains largely unknown. In this study, we observed that RNA N1-methyladenosine-generating methyltransferase TRMT6-TRMT61A complex is increased in aged murine HSCs due to aging-declined CRL4DCAF1-mediated ubiquitination degradation signaling. Unexpectedly, no difference of tRNA N1-methyladenosine methylome is observed between young and aged hematopoietic stem and progenitor cells, suggesting a noncanonical role of the TRMT6-TRMT61A complex in the HSC aging process. Further investigation revealed that enforced TRMT6-TRMT61A impairs HSCs through 3'-tiRNA-Leu-CAG and subsequent RIPK1-RIPK3-MLKL-mediated necroptosis cascade. Deficiency of necroptosis ameliorates the self-renewal capacity of HSCs and counters the physiologically deleterious effect of enforced TRMT6-TRMT61A on HSCs. Together, our work uncovers a nonclassical role for the TRMT6-TRMT61A complex in HSC aging and highlights a therapeutic target.

New advisory board members speak on challenges and opportunities in chemical biology.

Cell Chemical Biology has recently added several advisory board members from China in an effort to better represent our authorship and readership. In this Voices piece, a few of the new members introduce themselves, give their perspective on challenges and opportunities in chemical biology, and discuss how they plan to contribute to the field through their new position.

Multi-omics for COVID-19: driving development of therapeutics and vaccines.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has raised global concern for public health and economy. The development of therapeutics and vaccines to combat this virus is continuously progressing. Multi-omics approaches, including genomics, transcriptomics, proteomics, metabolomics, epigenomics and metallomics, have helped understand the structural and molecular features of the virus, thereby assisting in the design of potential therapeutics and accelerating vaccine development for COVID-19. Here, we provide an up-to-date overview of the latest applications of multi-omics technologies in strategies addressing COVID-19, in order to provide suggestions towards the development of highly effective knowledge-based therapeutics and vaccines.

Quantifying m6A and Ψ Modifications in the Transcriptome via Chemical-Assisted Approaches.

Since the discovery of the first chemically modified RNA nucleotide in 1951, more than 170 types of chemical modifications have been characterized in RNA so far. Since the discovery of the reversible and dynamic nature of N6-methyladenosine (m6A) in mRNA modification, researchers have identified about ten modifications in eukaryotic mRNA; together with modifications on the noncoding RNAs, the term "epitranscriptome" has been coined to describe the ensemble of various chemical RNA modifications. The past decade has witnessed the discovery of many novel molecular functions of mRNA modifications, demonstrating their crucial roles in gene expression regulation. As the most abundant modifications in mRNA, the study of m6A and Ψ has been facilitated by innovative high-throughput sequencing technologies, which can be based on antibodies, enzymes, or novel chemistry. Among them, chemical-assisted methods utilize selective chemistry that can discriminate modified versus unmodified nucleotides, enabling the transcriptome-wide mapping of m6A and Ψ modifications and functional studies.Our group has developed several sequencing technologies to investigate these epitranscriptomic marks including m6A, Ψ, m1A, and m6Am. Among them, we have recently developed two methods for absolute quantification of m6A and Ψ in the transcriptome based on chemical reactivity to distinguish and measure the two modifications. In GLORI, we used glyoxal and nitrite to mediate efficient deamination of regular adenosine, while m6A remained unaffected, thereby enabling efficient and unbiased detection of single-base resolution and absolute quantification of m6A modification. In CeU-seq and PRAISE, we used different chemistry to achieve selective labeling and detection of transcriptome-wide Ψ. CeU-seq is based on an azido-derivatized carbodiimide compound, while PRAISE utilizes the unique activity of bisulfite to Ψ. PRAISE results in the formation of ring-opening Ψ-bisulfite adduct and quantitatively detects Ψ as 1-2 nt deletion signatures during sequencing. The resulting base-resolution and stoichiometric information expanded our understanding to the profiles of RNA modifications in the transcriptome. In particular, the quantitative information on RNA methylome is critical for characterizing the dynamic and reversible nature of RNA modifications, for instance, under environmental stress or during development. Additionally, base-resolution and stoichiometric information can greatly facilitate the analysis and characterization of functional modification sites that are important for gene expression regulation, especially when one modification type may have multiple or even opposing functions within a specific transcript. Together, the quantitative profiling methods provide the modification stoichiometry information, which is critical to study the regulatory roles of RNA modifications.In this Account, we will focus on the quantitative sequencing technologies of m6A and Ψ developed in our group, review recent advances in chemical-assisted reactions for m6A and Ψ detection, and discuss the challenges and future opportunities of transcriptome-wide mapping technologies for RNA modifications.

Regulation and functions of non-m6A mRNA modifications.

Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant internal (non-cap) mRNA modification is N6-methyladenosine (m6A), the study of which has revolutionized our understanding of post-transcriptional gene regulation. In addition, numerous other mRNA modifications are gaining great attention because of their major roles in RNA metabolism, immunity, development and disease. In this Review, we focus on the regulation and function of non-m6A modifications in eukaryotic mRNA, including pseudouridine (Ψ), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), inosine, 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 2'-O-methylated nucleotide (Nm) and internal N7-methylguanosine (m7G). We highlight their regulation, distribution, stoichiometry and known roles in mRNA metabolism, such as mRNA stability, translation, splicing and export. We also discuss their biological consequences in physiological and pathological processes. In addition, we cover research techniques to further study the non-m6A mRNA modifications and discuss their potential future applications.

Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors.

Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.