Chemical Modification Of RNA Research Articles

N 1-methyladenosine (m1A) RNA modification: the key to ribosome control.

RNA displays diverse functions in living cells. The presence of various chemical modifications of RNA mediated by enzymes is one of the factors that impart such functional diversity to RNA. Among more than 100 types of RNA modification, N1-methyladenosine (m1A) is found mainly in tRNA and rRNA of many living organisms and is known to be deeply implicated in the topology or function of the two classes of RNA. In this commentary article, we would like to deal with the functional significance of m1A in RNA, and also to describe one methyltransferase installing m1A in a large subunit rRNA, whose orthologue in Caenorhabditis elegans was discovered recently and was reported in this journal.

RF-PseU: A Random Forest Predictor for RNA Pseudouridine Sites.

One of the ubiquitous chemical modifications in RNA, pseudouridine modification is crucial for various cellular biological and physiological processes. To gain more insight into the functional mechanisms involved, it is of fundamental importance to precisely identify pseudouridine sites in RNA. Several useful machine learning approaches have become available recently, with the increasing progress of next-generation sequencing technology; however, existing methods cannot predict sites with high accuracy. Thus, a more accurate predictor is required. In this study, a random forest-based predictor named RF-PseU is proposed for prediction of pseudouridylation sites. To optimize feature representation and obtain a better model, the light gradient boosting machine algorithm and incremental feature selection strategy were used to select the optimum feature space vector for training the random forest model RF-PseU. Compared with previous state-of-the-art predictors, the results on the same benchmark data sets of three species demonstrate that RF-PseU performs better overall. The integrated average leave-one-out cross-validation and independent testing accuracy scores were 71.4% and 74.7%, respectively, representing increments of 3.63% and 4.77% versus the best existing predictor. Moreover, the final RF-PseU model for prediction was built on leave-one-out cross-validation and provides a reliable and robust tool for identifying pseudouridine sites. A web server with a user-friendly interface is accessible at http://148.70.81.170:10228/rfpseu.

Abstract BS1-2: Genome regulation by long noncoding RNA genes

Abstract The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. I will discuss genome-scale strategies to discover and characterize lncRNAs, including RNA chemical modifications and RNA structures. LncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and target these enzymatic activities to appropriate locations in the genome. Long noncoding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. A new emerging theme is that DNA elements that mediate lncRNA expression can also have powerful effects in controlling chromatin neighborhoods, which alter oncogene expression and activity. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs in human diseases. Citation Format: HY Chang. Genome regulation by long noncoding RNA genes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BS1-2.

Writers, readers and erasers of RNA modifications in cancer.

Although cancer was originally considered a disease driven only by genetic mutations, it has now been proven that it is also an epigenetic disease driven by DNA hypermethylation-associated silencing of tumor suppressor genes and aberrant histone modifications. Very recently, a third component has emerged: the so-called epitranscriptome understood as the chemical modifications of RNA that regulate and alter the activity of RNA molecules. In this regard, the study of genetic and epigenetic disruption of the RNA-modifying proteins is gaining momentum in advancing our understanding of cancer biology. Furthermore, the development of epitranscriptomic anticancer drugs could lead to new promising and unexpected therapeutic strategies for oncology in the coming years.

RNA and CRISPR Interferences: Past, Present, and Future Perspectives.

RNA interference (RNAi), a natural gene silencing process, is a widely used technique in basic research, preclinical studies, and drug development strategies. Although the technique has great potential to generate new human therapiesand treat undruggable diseases, the clinical application of RNAi is still challenging primarily because of the delivery problem and potential off-target effects. Over the past two decades, great efforts have been undertaken to develop delivery agents and chemical modifications to overcome these challenges. Such advances in RNA delivery and chemical modifications have benefited researchers who are developing gene-editing therapies based on CRISPR-Cas9, an RNA-guided endonuclease, which is already having a major impact on biology and medicine. Here, I review the discovery of these two interference tools, identify the technical challenges yet to be overcome and provide some perspectives on how these two RNA-based technologies can be harnessed to treat human diseases.

N <sup>6</sup>-Methyladenosine Modification of LncRNA <i>Pvt1</i> Governs Epidermal Stemness

Dynamic chemical modifications of RNA represent novel and fundamental mechanisms that regulate stemness and tissue homeostasis. Rejuvenation and wound repair of mammalian skin are sustained by epidermal progenitor cells, which are localized within the basal layer of the skin epidermis. N6-methyladenosine (m6A) is one of the most abundant modifications found in eukaryotic mRNA and lncRNA (long non-coding RNA). In this report, we survey changes of m6A RNA methylomes upon epidermal differentiation, and identify Pvt1, a lncRNA whose m6A modification is critically involved in sustaining stemness of epidermal progenitor cells. With genome-editing and a mouse genetics approach, we show that ablation of m6A methyltransferase or Pvt1 impairs the self-renewal and wound healing capability of skin. Mechanistically, methylation of Pvt1 transcripts enhances its interaction with MYC and stabilizes the MYC protein in epidermal progenitor cells. Our study presents a global view of epitranscriptomic dynamics that occur during epidermal differentiation and identifies the m6A modification of Pvt1 as a key signaling event involved in skin tissue homeostasis and wound repair.

Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages.

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3'-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5'-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.

ADAR1 Safeguards MET by Suppressing ER Stress Through RNA Editing in Promoting Reprogramming

RNA chemical modifications are intricately linked to transcriptome structural and functional diversity. Whereas somatic cell reprogramming involves a profound rewiring of epigenetic and transcriptomic landscapes, how RNA modifications contribute to overriding somatic cell identity is incompletely understood. Here we demonstrate that loss of Adenosine-to-Inosine (A-to-I) editing of RNA by ADAR1 hampers mesenchymal-to-epithelial transition (MET) and impedes induced pluripotent stem cell (iPSC) formation. Absence of RNA editing leads to aberrant innate immune response (IIR) expression programs, which unleash endoplasmic reticulum (ER) stress and hinder epithelial fate acquisition. Using chemical and genetic approaches, we show a dual role for A-to-I editing in preventing MDA5-dependent activation of ER stress and ensuring proper double strand RNA (dsRNA) compartmentalization for PERK activation. Thus, our study establishes a critical role for A-to-I RNA modification in cell fate transitions during reprogramming, which delineates a novel regulatory layer underlying MET control for efficient reprogramming.

Interplay Between N 6-Methyladenosine (m6A) and Non-coding RNAs in Cell Development and Cancer.

RNA chemical modifications in coding and non-coding RNAs have been known for decades. They are generally installed by specific enzymes and, in some cases, can be read and erased by other specific proteins. The impact of RNA chemical modifications on gene expression regulation and the reversible nature of some of these modifications led to the birth of the word epitranscriptomics, in analogy with the changes that occur on DNA and histones. Among more than 100 different modifications identified so far, most of the epitranscriptomics studies focused on the N6-methyladenosine (m6A), which is the more abundant internal modification in protein coding RNAs. m6A can control several pathways of gene expression, including spicing, export, stability, and translation. In this review, we describe the interplay between m6A and non-coding RNAs, in particular microRNAs and lncRNAs, with examples of its role in gene expression regulation. Finally, we discuss its relevance in cell development and disease.

Optimization of RNA extraction from laser captured microdissected glomeruli from formalin-fixed paraffin-embedded mouse kidney samples for Nanostring analysis.

Optimized protocols for the microdissection of specific areas from archival tissues and the subsequent RNA analysis are needed but challenging due to RNA degradation and chemical modifications. The aim of this study was to present the most appropriate protocol for utilizing mouse FFPE kidney for laser capture microdissection and Nanostring gene expression analysis. We evaluated different section thicknesses (3, 5, 10 μm), 2 RNA extraction kits (Qiagen and Roche) and different H&E staining methods to optimize microdissection and RNA extraction from glomeruli and cortical tubules samples from FFPE mouse kidney. RNA quality and quantity were assessed via Nanodrop and Qubit. The protocol providing the best results consisted of 5 μm sections, a shorter protocol for H&E staining, and RNA extracted with the Roche kit. Higher Nanostring gene counts and lower qPCR cT significantly correlated with RNA concentrations measured with the Qubit, but not with measures obtained with the Nanodrop. The Nanostring data showed that none of the genes included in the panel was differentially expressed in the cortical tubule compartment compared to the whole kidney. However, 25 genes were differentially expressed in the glomerular compartment compared to the whole kidney. Our data showed that sufficient RNA can be extracted from small compartments like mouse renal glomeruli from archival FFPE tissue, and that whole kidney analysis does not accurately represent the transcriptome state of the glomeruli, which comprise only a small proportion of the overall kidney volume.

N6-Methyladenosine (m6A): A Promising New Molecular Target in Acute Myeloid Leukemia.

Recent studies have uncovered an important role for RNA modifications in gene expression regulation, which led to the birth of the epitranscriptomics field. It is now acknowledged that RNA modifiers play a crucial role in the control of differentiation of stem and progenitor cells and that changes in their levels are a relevant feature of different types of cancer. To date, among more than 160 different RNA chemical modifications, the more relevant in cancer biology is the reversible and dynamic N6-methylation of adenosine, yielding N6-methyladenosine (m6A). m6A is the more abundant internal modification in mRNA, regulating the expression of the latter at different levels, from maturation to translation. Here, we will describe the emerging role of m6A modification in acute myeloid leukemia (AML), which, among first, has demonstrated how mis-regulation of the m6A modifying system can contribute to the development and progression of cancer. Moreover, we will discuss how AML is paving the way to the development of new therapeutic options based on the inhibition of m6A deposition.

Chemical methods for the modification of RNA.

RNA is often considered as being the vector for the transmission of genetic information from DNA to the protein synthesis machinery. However, besides translation RNA participates in a broad variety of fundamental biological roles such as gene expression and regulation, protein synthesis, and even catalysis of chemical reactions. This variety of function combined with intricate three-dimensional structures and the discovery of over 100 chemical modifications in natural RNAs require chemical methods for the modification of RNAs in order to investigate their mechanism, location, and exact biological roles. In addition, numerous RNA-based tools such as ribozymes, aptamers, or therapeutic oligonucleotides require the presence of additional chemical functionalities to strengthen the nucleosidic backbone against degradation or enhance the desired catalytic or binding properties. Herein, the two main methods for the chemical modification of RNA are presented: solid-phase synthesis using phosphoramidite precursors and the enzymatic polymerization of nucleoside triphosphates. The different synthetic and biochemical steps required for each method are carefully described and recent examples of practical applications based on these two methods are discussed.

Discovery of Small Molecules that Activate RNA Methylation through Cooperative Binding to the METTL3-14-WTAP Complex Active Site

Chemical modifications of RNA provide an additional, epitranscriptomic, level of control over cellular functions. N-6-methylated adenosines (m6As) are found in several types of RNA, and their amounts are regulated by methyltransferases and demethylases. One of the most important enzymes catalyzing generation of m6A on mRNA is the trimer N-6-methyltransferase METTL3-14-WTAP complex. Its activity has been linked to such critical biological processes as cell differentiation, proliferation, and death. We used in silico-based discovery to identify small-molecule ligands that bind to METTL3-14-WTAP and determined experimentally their binding affinity and kinetics, as well as their effect on enzymatic function. We show that these ligands serve as activators of the METTL3-14-WTAP complex.

Factors affecting RNA quantification from tissue long-term stored in formalin

IntroductionFFPE samples represent a rich pool of tissue samples for retrospective analyses of mRNA and miRNA analyses. However, the initial formalin fixation introduces a chemical modification of RNA and causes its degradation, therefore, a longer storage of tissue in formalin is predicted to render ribonucleic acids lost for isolation and subsequent analyses. MethodsHerein, we tested the impact of several factors on isolation of total RNA and detection with RT-qPCR from mouse liver tissue stored for over two years in formalin at room temperature. ResultsThe incubation of tissue in TAE buffer before RNA isolation yielded higher concentration and purity of RNA and also improved subsequent analyses. Using gene-specific primers for cDNA synthesis and shorter PCR products (under 70 bp) significantly improved RT-qPCR analyses. We also compared the level of expression and differential expression of mRNA and miRNA with matched fresh frozen liver tissue, and observed similar changes in differential expression of selected mRNA and miRNA as in matched fresh frozen tissue. DiscussionConclusion is that adjustments of the protocol can substantially improve analyses of mRNA and miRNA from tissues stored in formalin for longer time.

Targeted m6A reader proteins to study the epitranscriptome.

Posttranscriptional regulation of RNA has emerged as an important regulator of genetic information flow in eukaryotic systems. In particular, chemical modifications of RNA have recently been established as key regulatory marks that affect the lifetime, location, trafficking, and function of messenger RNA (mRNA). In mammalian systems, N6-methyladenosine (m6A) is the most prevalent mRNA modification, and the writer, eraser, and reader proteins that install, remove, or recognize m6A have been rapidly uncovered and studied at the whole cell level. Understanding the effects of specific m6A modifications and their regulation at the single transcript level is the key next step to understanding the mechanism and consequences of epitranscriptomic regulation. We recently developed programmable m6A reader proteins to study the effects of epitranscriptomic regulatory factors at individual RNA transcripts. In this chapter, we discuss the application of targeted m6A readers to study RNA regulation at single endogenous sites. We briefly introduce what is currently known about the N6-methyltranscriptome and the Cas13 RNA-targeting family of proteins before detailing our protocol to study RNA modifications with targeted reader proteins.

Improved yield of rhEPO in CHO cells with synthetic 5' UTR.

The impact of local structure on mRNA translation is not well-defined pertaining to the 5' UTR. Reports suggest structural remodelling of the 5' UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5' UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5' UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer.

Visualizing the Role of 2'-OH rRNA Methylations in the Human Ribosome Structure.

Chemical modifications of RNA have recently gained new attention in biological sciences. They occur notably on messenger RNA (mRNA) and ribosomal RNA (rRNA) and are important for various cellular functions, but their molecular mechanism of action is yet to be understood in detail. Ribosomes are large ribonucleoprotein assemblies, which synthesize proteins in all organisms. Human ribosomes, for example, carry more than 200 modified nucleotides, which are introduced during biogenesis. Chemically modified nucleotides may appear to be only scarcely different from canonical nucleotides, but modifications such as methylations can in fact modulate their chemical and topological properties in the RNA and alter or modulate the overall translation efficiency of the ribosomes resulting in dysfunction of the translation machinery. Recent functional analysis and high-resolution ribosome structures have revealed a large repertoire of modification sites comprising different modification types. In this review, we focus on 2′-O-methylations (2′-O-Me) and discuss the structural insights gained through our recent cryo electron microscopy (cryo-EM) high-resolution structural analysis of the human ribosome, such as their locations and their influence on the secondary and tertiary structures of human rRNAs. The detailed analysis presented here reveals that ribose conformations of the rRNA backbone differ when the 2′-OH hydroxyl position is methylated, with 3′-endo conformations being the default and the 2′-endo conformations being characteristic in that the associated base is flipped-out. We compare currently known 2′-O-Me sites in human rRNAs evaluated using RiboMethSeq and cryo-EM structural analysis and discuss their involvement in several human diseases.

Landscape of the complete RNA chemical modifications in the human 80S ribosome.

During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

PEA: an integrated R toolkit for plant epitranscriptome analysis.

The epitranscriptome, also known as chemical modifications of RNA (CMRs), is a newly discovered layer of gene regulation, the biological importance of which emerged through analysis of only a small fraction of CMRs detected by high-throughput sequencing technologies. Understanding of the epitranscriptome is hampered by the absence of computational tools for the systematic analysis of epitranscriptome sequencing data. In addition, no tools have yet been designed for accurate prediction of CMRs in plants, or to extend epitranscriptome analysis from a fraction of the transcriptome to its entirety. Here, we introduce PEA, an integrated R toolkit to facilitate the analysis of plant epitranscriptome data. The PEA toolkit contains a comprehensive collection of functions required for read mapping, CMR calling, motif scanning and discovery and gene functional enrichment analysis. PEA also takes advantage of machine learning (ML) technologies for transcriptome-scale CMR prediction, with high prediction accuracy, using the Positive Samples Only Learning algorithm, which addresses the two-class classification problem by using only positive samples (CMRs), in the absence of negative samples (non-CMRs). Hence PEA is a versatile epitranscriptome analysis pipeline covering CMR calling, prediction and annotation and we describe its application to predict N6-methyladenosine (m6A) modifications in Arabidopsis thaliana. Experimental results demonstrate that the toolkit achieved 71.6% sensitivity and 73.7% specificity, which is superior to existing m6A predictors. PEA is potentially broadly applicable to the in-depth study of epitranscriptomics. PEA Docker image is available at https://hub.docker.com/r/malab/pea, source codes and user manual are available at https://github.com/cma2015/PEA. Supplementary data are available at Bioinformatics online.

RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.

METTL3 is frequently up-regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A-YTHDF2-dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254-2270).