Chemical Modification Of RNA Research Articles

Mass Spectrometry-Based Proteomics for Assessing Epitranscriptomic Regulations.

Epitranscriptomics is a rapidly evolving field that explores chemical modifications in RNA and how they contribute to dynamic and reversible regulations of gene expression. These modifications, for example, N6-methyladenosine (m6A), are crucial in various RNA metabolic processes, including splicing, stability, subcellular localization, and translation efficiency of mRNAs. Mass spectrometry-based proteomics has become an indispensable tool in unraveling the complexities of epitranscriptomics, offering high-throughput, precise protein identification, and accurate quantification of differential protein expression. Over the past two decades, advances in mass spectrometry, including the improvement of high-resolution mass spectrometers and innovative sample preparation methods, have allowed researchers to perform in-depth analyses of epitranscriptomic regulations. This review focuses on the applications of bottom-up proteomics in the field of epitranscriptomics, particularly in identifying and quantifying epitranscriptomic reader, writer, and eraser (RWE) proteins and in characterizing their functions, posttranslational modifications, and interactions with other proteins. Together, by leveraging modern proteomics, researchers can gain deep insights into the intricate regulatory networks of RNA modifications, advancing fundamental biology, and fostering potential therapeutic applications.

Post-Translational Modifications of RNA-Modifying Proteins in Cellular Dynamics and Disease Progression.

RNA-modifying proteins, classified as "writers," "erasers," and "readers," dynamically modulate RNA by adding, removing, or interpreting chemical groups, thereby influencing RNA stability, functionality, and interactions. To date, over 170 distinct RNA chemical modifications and more than 100 RNA-modifying enzymes have been identified, with ongoing research expanding these numbers. Although significant progress has been made in understanding RNA modification, the regulatory mechanisms that govern RNA-modifying proteins themselves remain insufficiently explored. Post-translational modifications (PTMs) such as phosphorylation, ubiquitination, and acetylation are crucial in modulating the function and behavior of these proteins. However, the full extent of PTM influence on RNA-modifying proteins and their role in disease development remains to be fully elucidated. This review addresses these gaps by offering a comprehensive analysis of the roles PTMs play in regulating RNA-modifying proteins. Mechanistic insights are provided into how these modifications alter biological processes, contribute to cellular function, and drive disease progression. In addition, the current research landscape is examined, highlighting the therapeutic potential of targeting PTMs on RNA-modifying proteins for precision medicine. By advancing understanding of these regulatory networks, this review seeks to facilitate the development of more effective therapeutic strategies and inspire future research in the critical area of PTMs in RNA-modifying proteins.

MRNA 5-methylcytosine in Eimeria tenella oocysts: An abundant post-transcriptional modification associated with broad-ranging biological processes

Eimeria tenella is the major causative agent of chicken coccidiosis. 5-Methylcytosine (m5C) is a type of RNA chemical modifications reported to regulate diverse biological processes. However, the distribution and biological functions of m5C in E. tenella mRNAs are yet to be known. Herein, we report transcriptome-wide profiling of mRNA m5C in E. tenella by employing m5C RNA immunoprecipitation followed by a deep-sequencing approach (m5C-RIP-seq). Our data showed that m5C peaks were distributed across the whole mRNA body. Compared with unsporulated oocysts, there were 2813 hypermethylated and 1850 hypomethylated m5C peaks in sporulated oocysts. Generally, a positive correlation between m5C modification and gene expression levels was observed. The mRNA sequencing (RNA-seq) and m5C-RIP-seq data were consistent with the results of the quantitative reverse transcription PCR (RT-qPCR) and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR), respectively. Gene Ontology (GO) and pathway enrichment analysis predicated diverse biological functions and pathways, including microtubule motor activity, helicase activity, cGMP-PKG signaling pathway, aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis, and spliceosome. Meanwhile, stage-specific gene expression signatures of m5C-related regulators were observed. Altogether, our findings reveal the transcriptional significance of m5C modification in E. tenella oocysts, providing resources and clues for further in-depth research.

Sia-m7G: Predicting m7G Sites through the Siamese Neural Network with an Attention Mechanism

Background: The chemical modification of RNA plays a crucial role in many biological processes. N7-methylguanosine (m7G), being one of the most important epigenetic modifications, plays an important role in gene expression, processing metabolism, and protein synthesis. Detecting the exact location of m7G sites in the transcriptome is key to understanding their relevant mechanism in gene expression. On the basis of experimentally validated data, several machine learning or deep learning tools have been designed to identify internal m7G sites and have shown advantages over traditional experimental methods in terms of speed, cost-effectiveness and robustness. Aims: In this study, we aim to develop a computational model to help predict the exact location of m7G sites in humans. Objective: Simple and advanced encoding methods and deep learning networks are designed to achieve excellent m7G prediction efficiently. Methods: Three types of feature extractions and six classification algorithms were tested to identify m7G sites. Our final model, named Sia-m7G, adopts one-hot encoding and a delicate Siamese neural network with an attention mechanism. In addition, multiple 10-fold cross-validation tests were conducted to evaluate our predictor. Results: Sia-m7G achieved the highest sensitivity, specificity and accuracy on 10-fold crossvalidation tests compared with the other six m7G predictors. Nucleotide preference and model visualization analyses were conducted to strengthen the interpretability of Sia-m7G and provide a further understanding of m7G site fragments in genomic sequences. Conclusion: Sia-m7G has significant advantages over other classifiers and predictors, which proves the superiority of the Siamese neural network algorithm in identifying m7G sites.

RNA modifications in insects.

More than 100 RNA chemical modifications to cellular RNA have been identified. N 6-methyladenosine (m6A) is the most prevalent modification of mRNA. RNA modifications have recently attracted significant attention due to their critical role in regulating mRNA processing and metabolism. tRNA and rRNA rank among the most heavily modified RNAs, and their modifications are essential for maintaining their structure and function. With our advanced understanding of RNA modifications, increasing evidence suggests RNA modifications are important in regulating various aspects of insect life. In this review, we will summarize recent studies investigating the impact of RNA modifications in insects, particularly highlighting the role of m6A in insect development, reproduction, and adaptation to the environment.

Synthesis and Properties of RNA Modified with Thioamide Internucleoside Linkage.

Recent success of RNA therapeutics has reinvigorated interest in chemical modifications of RNA. As exemplified by the phosphorothioates, modifications of sugar-phosphate backbone have been remarkably impactful but relatively underexplored in therapeutic RNAs. The present study reports synthesis, thermal stability, and RNA interference activity of RNAs modified with thioamide linkages. Compared to the previously studied amide-modified RNA, thioamide linkages strongly destabilized a short self-complementary RNA model duplex. However, in short interfering RNAs amides and thioamides had a similar effect on duplex stability and target RNA cleavage activity and specificity. Hence, the thioamide may be added to the toolbox of chemical biologist as a useful backbone modification well tolerated by the RNA interference machinery.

Exploring the impact of m6A modification on immune diseases: mechanisms and therapeutic implication.

N6-methyladenosine (m6A) is a chemical modification of RNA and has become a widely discussed topic among scientific researchers in recent years. It is distributed in various organisms, including eukaryotes and bacteria. It has been found that m6A is composed of writers, erasers and readers and is involved in biological functions such as splicing, transport and translation of RNA. The balance of the human immune microenvironment is important for human health abnormalities. Increasing studies have found that m6A affects the development of immune diseases such as inflammatory enteritis and systemic lupus erythematosus (SLE) by participating in the homeostatic regulation of the immune microenvironment in vivo. In this manuscript, we introduce the composition, biological function, regulation of m6A in the immune microenvironment and its progression in various immune diseases, providing new targets and directions for the treatment of immune diseases in clinical practice.

Promoted Read-through and Mutation Against Pseudouridine-CMC by an Evolved Reverse Transcriptase.

Pseudouridine (Ψ) is an abundant RNA chemical modification that can play critical roles in the biological functions of RNA, and RNA-therapeutic applications. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. The N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) can selectively label Ψ in RNA by forming the CMC-Ψ adduct. Here we report that an evolved reverse transcriptase ("RT-1306") shows promoted read-through and mutation against the CMC-Ψ. The mutation signature can resolve the occurrence of Ψs within UU-containing sequences. We developed "Mut-Ψ-seq" utilizing CMC and RT-1306 for transcriptome-wide mapping of Ψ at base-resolution. The mutation signatures robustly identify reported Ψs in human rRNAs via the ROC analysis, and elongated CMC reaction duration increases the detection sensitivity of Ψ. We report a high-confidence list of Ψ sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of Ψ in UU-containing sequences, revealing diverse occurrence of Ψs in such sequences. This work provides new methods and datasets for biological research of Ψ, and demonstrates the potential of combining the reverse transcriptase engineering and selective chemical labeling to expand the toolkit for RNA chemical modifications studies.

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding.

This comprehensive Review delves into the chemical principles governing RNA-mediated crowding events, commonly referred to as granules or biological condensates. We explore the pivotal role played by RNA sequence, structure, and chemical modifications in these processes, uncovering their correlation with crowding phenomena under physiological conditions. Additionally, we investigate instances where crowding deviates from its intended function, leading to pathological consequences. By deepening our understanding of the delicate balance that governs molecular crowding driven by RNA and its implications for cellular homeostasis, we aim to shed light on this intriguing area of research. Our exploration extends to the methodologies employed to decipher the composition and structural intricacies of RNA granules, offering a comprehensive overview of the techniques used to characterize them, including relevant computational approaches. Through two detailed examples highlighting the significance of noncoding RNAs, NEAT1 and XIST, in the formation of phase-separated assemblies and their influence on the cellular landscape, we emphasize their crucial role in cellular organization and function. By elucidating the chemical underpinnings of RNA-mediated molecular crowding, investigating the role of modifications, structures, and composition of RNA granules, and exploring both physiological and aberrant phase separation phenomena, this Review provides a multifaceted understanding of the intriguing world of RNA-mediated biological condensates.

MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64–106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

Abstract 4417: Development of biochemical screening assays to facilitate drug discovery in RNA m6A modification regulators

Abstract Over the recent years, knowledge of RNA modifications and their regulation has expanded extensively, providing novel insights and strategies to explore potential therapeutics for pathogenesis of various diseases, including cancer. Several chemical modifications in RNA have been identified so far, among these N6-methyladenosine (m6A) modification is the most abundant and well-studied epitranscriptomic marker found in mRNA and long noncoding RNA. Abnormal m6A expression is proven to be associated with tumorigenesis, cancer stemness and drug resistance of cancers. Methylation in 6-Adenosin of RNA is a dynamic and reversible process tightly regulated by its writer, m6A methyltransferase complex and erasers, FTO and ALKBH5. m6A methyltransferase complex contains METTL3-METTL14-WTAP as core components. METTL3 is the catalytic component, which is activated by heterodimer formation with METTL14. High METTL3 expression is found in several cancers such as breast, lung, liver, gastric, colorectal, AML and a METTL3 catalytic inhibitor is found to delay AML progression in mouse models. Protein that binds RNA m6A modification to execute it signaling are known as m6A readers. Readers are categorized into three main classes, YTH domain proteins, IGF2 mRNA-binding proteins and heterogeneous nuclear ribonucleoproteins. YTH family readers are found to have oncogenic roles in several cancers including AML, breast, lung, CRC and glioblastoma. Recent studies suggest inhibition of m6A binding of individual YTH family proteins is a promising therapeutic strategy however, potent inhibitors are yet to be identified. This poster summarizes current assays we have developed to facilitate cancer therapeutic discovery in m6A related protein targets. We present development and validation of an assay suitable identify in METTL3 catalytic inhibitors using our proprietary hotspot technology. We also show development of HTRF based biochemical assays for all five YTH family protein YTHDF1-3 and YTHDC1-2, to screen for molecules that disrupt protein-m6A interaction. We further study the selectivity of an FDA approved drug Tegaserod, a reported YTHDF1 inhibitor by a structure based virtual screening, between YTH family proteins. Citation Format: Safnas F. AbdulSalam, Sung Won Oh, Brenna P. Lee, Joseph J. Ferry, John R. Ries, Kurumi Y. Horiuchi. Development of biochemical screening assays to facilitate drug discovery in RNA m6A modification regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4417.

Sequencing, Physiological Regulation, and Representative Disease Research Progress of RNA m6A Modification

AbstractTo date, more than 150 chemical modifications have been disclosed in different RNA species, which are employed to diversify the structure and function of RNA in living organisms. The N 6-methyladenosine (m6A) modification, which is found in the adenosine N 6 site of RNA, has been demonstrated to be the most heavy modification in the mRNA in cells. Moreover, the m6A modification in mRNAs of mammalian and other eukaryotic cells is highly conserved and mandatorily encoded. Increasing evidence indicates that the m6A modification plays a pivotal role in gene-expression regulation and cell-fate decisions. Here, we summarize the most recent m6A-sequencing technology, as well as the molecular mechanism underlying its occurrence, development, and potential use as a target for the treatment of human diseases. Furthermore, our review highlights other newly discovered chemical modifications of RNA that are associated with human disease, as well as their underlying molecular mechanisms. Thus, significant advancements have been made in qualitative/quantitative m6A detection and high-throughput sequencing, and research linking this RNA modification to disease. Efforts toward simplified and more accessible chemical/biological technologies that contribute to precision medicine are ongoing, to benefit society and patients alike.

Synthesis and Properties of RNA Modified with Cationic Amine Internucleoside Linkage.

Chemical modifications of RNA are important tools for the development of RNA therapeutics. The present study reports a novel RNA backbone modification that replaces the negatively charged phosphate with a positively charged amine linkage. Despite being thermally destabilizing in RNA duplexes, the amine linkage caused a relatively modest decrease of activity of a modified short interfering RNA (siRNA). At position 2 of the guide strand, the amine modification strongly enhanced the specificity of siRNA while causing an ∼5-fold drop of on-target activity. These results support the future development of amines as cationic RNA modifications and novel tools to modulate protein-RNA interactions.

MeRIP-Seq for Identifying Stress-Responsive Transcriptome-Wide m6A Profiles in Plants.

Recent advancements in detection and mapping methods have enabled researchers to uncover the biological importance of RNA chemical modifications, which play a vital role in post-transcriptional gene regulation. Although numerous types of RNA modifications have been identified in higher eukaryotes, only a few have been extensively studied for their biological functions. Of these, N6-methyladenosine (m6A) is the most prevalent and important mRNA modification that influences various aspects of RNA metabolism, including mRNA stability, degradation, splicing, alternative polyadenylation, export, and localization, as well as translation. Thus, they have implications for a variety of biological processes, including growth, development, and stress responses. The m6A deposition or removal on transcripts is dynamic and is altered in response to internal and external cues. Because this mark can alter gene expression under stress conditions, it is essential to identify the transcripts that can acquire or lose this epitranscriptomic mark upon exposure to stress conditions. Here we describe a step-by-step protocol for identifying stress-responsive transcriptome-wide m6A changes using RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq).

WTAP-Mediated N6-Methyladenosine of RNAs Facilitate the Pathophysiology of Atopic Dermatitis

N6-methyladenosine (m6A) is the most abundant dynamic and reversible internal chemical modification of RNA in eukaryotic cells and is essential in multiple pathophysiological processes. However, it has not been reported in atopic dermatitis (AD). We used Arraystar m6A-mRNA epitranscriptomic microarray to screen for differentially expressed genes (DEGs) and their m6A levels, and m6A-related enzymes in patients with AD. We confirmed that the m6A RNA methyltransferase Wilms tumor 1-associated protein (WTAP) and two candidate DEGs (S100 calcium-binding protein A9 [S100A9] and serpin peptidase inhibitor, clade B (ovalbumin), member 3 [SERPINB3] were significantly upregulated in keratinocytes in public data and epidermal lesions of patients with AD. In vitro cell experiments confirmed that WTAP influenced the expression of the two candidate DEGs and promoted primary human epidermal keratinocytes (HEKs) proliferation while inhibiting HEKs differentiation. Furthermore, we showed that WTAP, S100A9, and SERPINB3 expression correlated with AD severity. Our findings revealed that WTAP-mediated m6A modification promoted the expression of S100A9 and SERPINB3 to aggravate HEK proliferation and dysdifferentiation contributing to the pathophysiological development of AD.

Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

Diadenosine Tetraphosphate (Ap4A) Serves as a 5′ RNA Cap in Mammalian Cells

AbstractThe recent expansion of the field of RNA chemical modifications has changed our understanding of post‐transcriptional gene regulation. Apart from internal nucleobase modifications, 7‐methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non‐canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non‐canonical RNA cap, diadenosine tetraphosphate (Ap4A), in human and rat cell lines. Ap4A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non‐canonical initiating nucleotide (NCIN). Using liquid chromatography‐mass spectrometry (LC–MS), we show that the amount of capped Ap4A‐RNA is independent of the cellular concentration of Ap4A. A decapping enzyme screen identifies two enzymes cleaving Ap4A‐RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4A‐RNA and show that although it is not translated, Ap4A‐RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

RNA Editing in Cancer Progression.

Coding and noncoding RNA molecules play their roles in ensuring cell function and tissue homeostasis in an ordered and systematic fashion. RNA chemical modifications can occur both at bases and ribose sugar, and, similarly to DNA and histone modifications, can be written, erased, and recognized by the corresponding enzymes, thus modulating RNA activities and fine-tuning gene expression programs. RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification in normal physiological processes. By altering the sequences of mRNAs, it makes them different from the corresponding genomic template. Hence, edited mRNAs can produce protein isoforms that are functionally different from the corresponding genome-encoded variants. Abnormalities in regulatory enzymes and changes in RNA-modification patterns are closely associated with the occurrence and development of various human diseases, including cancer. To date, the roles played by RNA modifications in cancer are gathering increasing interest. In this review, we focus on the role of RNA editing in cancer transformation and provide a new perspective on its impact on tumorigenesis, by regulating cell proliferation, differentiation, invasion, migration, stemness, metabolism, and drug resistance.

RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples.

Clinical tumor tissues that are preserved as formalin-fixed paraffin-embedded (FFPE) samples result in extensive cross-linking, fragmentation, and chemical modification of RNA, posing significant challenges for RNA-seq-based gene expression profiling. This study sought to define an optimal RNA-seq protocol for FFPE samples. We employed a common RNA extraction method and then compared RNA-seq library preparation protocols including RNAaccess, RiboZero and PolyA in terms of sequencing quality and concordance of gene expression using FFPE and case-matched fresh-frozen (FF) triple-negative breast cancer (TNBC) tissues. We found that RNAaccess, a method based on exome capture, produced the most concordant results. Applying RNAaccess to FFPE gastric cancer tissues, we established a minimum RNA DV200 requirement of 10% and a RNA input amount of 10ng that generated highly reproducible gene expression data. Lastly, we demonstrated that RNAaccess and NanoString platforms produced highly concordant expression profiles from FFPE samples for shared genes; however, RNA-seq may be preferred for clinical biomarker discovery work because of the broader coverage of the transcriptome. Taken together, these results support the selection of RNA-seq RNAaccess method for gene expression profiling of FFPE samples. The minimum requirements for RNA quality and input established here may allow for inclusion of clinical FFPE samples of sub-optimal quality in gene expression analyses and ultimately increasing the statistical power of such analyses.

Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives.

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.