RNA 5-methylcytosine Research Articles

RNAm5Cfinder: A Web-server for Predicting RNA 5-methylcytosine (m5C) Sites Based on Random Forest

5-methylcytosine (m5C) is a common nucleobase modification, and recent investigations have indicated its prevalence in cellular RNAs including mRNA, tRNA and rRNA. With the rapid accumulation of m5C sites data, it becomes not only feasible but also important to build an accurate model to predict m5C sites in silico. For this purpose, here, we developed a web-server named RNAm5Cfinder based on RNA sequence features and machine learning method to predict RNA m5C sites in eight tissue/cell types from mouse and human. We confirmed the accuracy and usefulness of RNAm5Cfinder by independent tests, and the results show that the comprehensive and cell-specific predictors could pinpoint the generic or tissue-specific m5C sites with the Area Under Curve (AUC) no less than 0.77 and 0.87, respectively. RNAm5Cfinder web-server is freely available at http://www.rnanut.net/rnam5cfinder.

Selectivity and Promiscuity in TET-Mediated Oxidation of 5-Methylcytosine in DNA and RNA.

Enzymes of the ten-eleven translocation (TET) family add diversity to the repertoire of nucleobase modifications by catalyzing the oxidation of 5-methylcytosine (5mC). TET enzymes were initially found to oxidize 5-methyl-2'-deoxycytidine in genomic DNA, yielding products that contribute to epigenetic regulation in mammalian cells, but have since been found to also oxidize 5-methylcytidine in RNA. Considering the different configurations of single-stranded (ss) and double-stranded (ds) DNA and RNA that coexist in a cell, defining the scope of TET's preferred activity and the mechanisms of substrate selectivity is critical to better understand the enzymes' biological functions. To this end, we have systematically examined the activity of human TET2 on DNA, RNA, and hybrid substrates in vitro. We found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA. Chimeric and hybrid substrates containing mixed DNA and RNA character helped reveal two main features by which the enzyme discriminates between substrates. First, the identity of the target nucleotide alone is the strongest reactivity determinant, with a preference for 5-methyldeoxycytidine, while both DNA or RNA are relatively tolerated on the rest of the target strand. Second, while a complementary strand is not required for activity, DNA is the preferred partner, and complementary RNA diminishes reactivity. Our biochemical analysis, complemented by molecular dynamics simulations, provides support for an active site optimally configured for dsDNA reactivity but permissive for various nucleic acid configurations, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.

Accurate RNA 5-methylcytosine site prediction based on heuristic physical-chemical properties reduction and classifier ensemble

RNA 5-methylcytosine (m5C) is an important post-transcriptional modification that plays an indispensable role in biological processes. The accurate identification of m5C sites from primary RNA sequences is especially useful for deeply understanding the mechanisms and functions of m5C. Due to the difficulty and expensive costs of identifying m5C sites with wet-lab techniques, developing fast and accurate machine-learning-based prediction methods is urgently needed. In this study, we proposed a new m5C site predictor, called M5C-HPCR, by introducing a novel heuristic nucleotide physicochemical property reduction (HPCR) algorithm and classifier ensemble. HPCR extracts multiple reducts of physical-chemical properties for encoding discriminative features, while the classifier ensemble is applied to integrate multiple base predictors, each of which is trained based on a separate reduct of the physical-chemical properties obtained from HPCR. Rigorous jackknife tests on two benchmark datasets demonstrate that M5C-HPCR outperforms state-of-the-art m5C site predictors, with the highest values of MCC (0.859) and AUC (0.962). We also implemented the webserver of M5C-HPCR, which is freely available at http://cslab.just.edu.cn:8080/M5C-HPCR/.

RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

Tet protein function during Drosophila development.

The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. Here, we characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. We show that Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, we report the function of Tet in ovarian morphogenesis. Together, our findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.

IRNAm5C-PseDNC: identifying RNA 5-methylcytosine sites by incorporating physical-chemical properties into pseudo dinucleotide composition.

Occurring at cytosine (C) of RNA, 5-methylcytosine (m5C) is an important post-transcriptional modification (PTCM). The modification plays significant roles in biological processes by regulating RNA metabolism in both eukaryotes and prokaryotes. It may also, however, cause cancers and other major diseases. Given an uncharacterized RNA sequence that contains many C residues, can we identify which one of them can be of m5C modification, and which one cannot? It is no doubt a crucial problem, particularly with the explosive growth of RNA sequences in the postgenomic age. Unfortunately, so far no user-friendly web-server whatsoever has been developed to address such a problem. To meet the increasingly high demand from most experimental scientists working in the area of drug development, we have developed a new predictor called iRNAm5C-PseDNC by incorporating ten types of physical-chemical properties into pseudo dinucleotide composition via the auto/cross-covariance approach. Rigorous jackknife tests show that its anticipated accuracy is quite high. For most experimental scientists’ convenience, a user-friendly web-server for the predictor has been provided at http://www.jci-bioinfo.cn/iRNAm5C-PseDNC along with a step-by-step user guide, by which users can easily obtain their desired results without the need to go through the complicated mathematical equations involved. It has not escaped our notice that the approach presented here can also be used to deal with many other problems in genome analysis.

Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs.

Posttranscriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification with diverse roles, such as regulating stress responses, stem cell proliferation, and RNA metabolism. Here, we used RNA bisulfite sequencing for transcriptome-wide quantitative mapping of m5C in the model plant Arabidopsis thaliana We discovered more than a thousand m5C sites in Arabidopsis mRNAs, long noncoding RNAs, and other noncoding RNAs across three tissue types (siliques, seedling shoots, and roots) and validated a number of these sites. Quantitative differences in methylated sites between these three tissues suggest tissue-specific regulation of m5C. Perturbing the RNA m5C methyltransferase TRM4B resulted in the loss of m5C sites on mRNAs and noncoding RNAs and reduced the stability of tRNAAsp(GTC) We also demonstrate the importance of m5C in plant development, as trm4b mutants have shorter primary roots than the wild type due to reduced cell division in the root apical meristem. In addition, trm4b mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50-nucleotide sequence flanking m5C C3349 in MAIGO5 mRNA is sufficient to confer methylation of a transgene reporter in Nicotiana benthamiana.

Post-transcriptional modifications in development and stem cells.

Cells adapt to their environment by linking external stimuli to an intricate network of transcriptional, post-transcriptional and translational processes. Among these, mechanisms that couple environmental cues to the regulation of protein translation are not well understood. Chemical modifications of RNA allow rapid cellular responses to external stimuli by modulating a wide range of fundamental biochemical properties and processes, including the stability, splicing and translation of messenger RNA. In this Review, we focus on the occurrence of N6-methyladenosine (m6A), 5-methylcytosine (m5C) and pseudouridine (Ψ) in RNA, and describe how these RNA modifications are implicated in regulating pluripotency, stem cell self-renewal and fate specification. Both post-transcriptional modifications and the enzymes that catalyse them modulate stem cell differentiation pathways and are essential for normal development.

Sequence-specific and Shape-selective RNA Recognition by the Human RNA 5-Methylcytosine Methyltransferase NSun6

Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.

Formation and determination of the oxidation products of 5-methylcytosine in RNA.

Similar to the reversible epigenetic modifications on DNA, dynamic RNA modifications were recently considered to constitute another realm for biological regulation in the form of "RNA epigenetics". 5-Methylcytosine (5-mC) has long been known to be present in RNA from all three kingdoms of life. However, the functions of 5-mC in RNA have not been fully understood, especially for the RNA demethylation mechanism. The discovery of 5-hydroxymethylcytosine (5-hmC) in RNA together with our recently reported 5-formylcytosine (5-foC) in RNA indicated that 5-mC in RNA may undergo the same cytosine oxidation demethylation pathway with generating intermediates 5-hmC, 5-foC, and 5-carboxylcytosine (5-caC) by ten-eleven translocation (Tet) proteins as that in DNA. However, endogenous 5-caC in RNA has not been observed so far. In the current study, we established a method using chemical labeling coupled with liquid chromatography-mass spectrometry analysis for the sensitive and simultaneous determination of the oxidative products of 5-mC. Our results demonstrated that the detection sensitivities of 5-mC, 5-hmC, 5-foC and 5-caC in RNA increased by 70-313 folds upon 2-bromo-1-(4-diethylaminophenyl)-ethanone (BDEPE) labeling. Using this method, we discovered the existence of 5-caC in the RNA of mammals. In addition, we found the 5-mC occurs in all RNA species including mRNA, 28S rRNA, 18S rRNA and small RNA (<200 nt). However, 5-hmC, 5-foC and 5-caC mainly occur in mRNA, and barely detected in other types of RNA. Furthermore, we found that the content of 5-hmC in the RNA of human colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC) tissues significantly decreased compared to tumor adjacent normal tissues, suggesting that 5-hmC in RNA may play certain functional roles in the regulation of cancer development and formation.

Guitar: An R/Bioconductor Package for Gene Annotation Guided Transcriptomic Analysis of RNA-Related Genomic Features.

Biological features, such as genes and transcription factor binding sites, are often denoted with genome-based coordinates as the genomic features. While genome-based representation is usually very effective in correlating various biological features, it can be tedious to examine the relationship between RNA-related genomic features and the landmarks of RNA transcripts with existing tools due to the difficulty in the conversion between genome-based coordinates and RNA-based coordinates. We developed here an open source Guitar R/Bioconductor package for sketching the transcriptomic view of RNA-related biological features represented by genome based coordinates. Internally, Guitar package extracts the standardized RNA coordinates with respect to the landmarks of RNA transcripts, with which hundreds of millions of RNA-related genomic features can then be efficiently analyzed within minutes. We demonstrated the usage of Guitar package in analyzing posttranscriptional RNA modifications (5-methylcytosine and N6-methyladenosine) derived from high-throughput sequencing approaches (MeRIP-Seq and RNA BS-Seq) and show that RNA 5-methylcytosine (m5C) is enriched in 5′UTR. The newly developed Guitar R/Bioconductor package achieves stable performance on the data tested and revealed novel biological insights. It will effectively facilitate the analysis of RNA methylation data and other RNA-related biological features in the future.

Identifying RNA 5-methylcytosine sites via pseudo nucleotide compositions.

RNA 5-methylcytosine (m5C) plays an important role in numerous biological processes. Accurate identification of the m5C site is helpful for a better understanding of its biological functions. However, the drawbacks of the experimental methods available preclude progress towards the identification of the m5C site. As an excellent complement to experimental techniques, computational methods will facilitate the identification of m5C. In the present study, a support vector machine based-method is proposed to identify m5C sites in Homo sapiens. In this method, RNA sequences are encoded using the pseudo dinucleotide composition in which three RNA physiochemical properties are incorporated. It was observed by the jackknife cross-validation that the overall success rate achieved by the proposed model is 90.42%. This result indicates that the proposed model holds the potential to become a useful tool for the identification of m5C sites.

Variable presence of 5-methylcytosine in commercial RNA and DNA

Nucleoside methylations and other nucleic acid modifications have recently encountered a surge in interest, prompted, among other things, by the detection of methylation and active demethylation of DNA and mRNA by similar mechanisms. In DNA, deoxycytidine methylation by Dnmt enzymes generates 5-methyldeoxycytidine,1 an important epigenetic mark that typically causes inactivation of transcription of the methylated promoter region. Recent exciting developments have shown that these marks are not concrete-cast, but can be actively removed by the oxidative action of TET enzymes,2 which generate, through a series of 2-electron oxidations, first hydroxymethylcytidine (hm5C), then formyldeoxycytidine (f5C),3 and finally carboxydeoxycytidine (ca5C), which may eventually regenerate deoxycytidine by decarboxylation. The apparent functional homolog in mRNA is m6A, which appears to reduce translation efficiency. Here, too, the methylation can be removed by TET-related enzymes generating first hydroxymethyladenosine (hm6A), then formyladenosine (f6A).4,5,6 Also, the presence of 5-methylcytidine in mRNA has been reported early on, and has recently raised renewed interest, although its function is as yet unclear and putative conversion into hydroxymethylcytidine is yet to be demonstrated.32 These developments are enhanced by the development of highly sensitive detection methods7,8 including the adaptation of the so-called bisulfite sequencing from DNA, where it is well established,9 to RNA, where its application has significantly contributed to the present high level of interest.10,11,12 However, bisulfite sequencing alone does not yield unassailable results13,14 and we have thus looked to expand the limits of detection of m5C in both DNA and RNA by LC-MS/MS. Here we report a straightforward regimen that provides values for the limit of quantification (LOQ) in the triple digit attomol range. Its application to presumed negative controls, namely synthetic oligonucleotides, surprisingly detected significant amounts of m5C in both types of synthetic nucleic acids.

Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae.

BackgroundPost-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification that regulates RNA metabolism and occurs in both eukaryotes and prokaryotes. Yet, to date, no transcriptome-wide identification of m5C sites has been undertaken in plants. Plants provide a unique comparative system for investigating the origin and evolution of m5C as they contain three different genomes, the nucleus, mitochondria and chloroplast. Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in non-coding ribosomal RNAs and transfer RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba.ResultsUsing the plant model Arabidopsis thaliana, we identified a total of 39 highly methylated m5C sites in predicted structural positions of nuclear tRNAs and 7 m5C sites in rRNAs from nuclear, chloroplast and mitochondrial transcriptomes. Both the nucleotide position and percent methylation of tRNAs and rRNAs m5C sites were conserved across all species analysed, from single celled algae N. oculata to multicellular plants. Interestingly the mitochondrial and chloroplast encoded tRNAs were devoid of m5C in A. thaliana and this is generally conserved across Plantae. This suggests independent evolution of organelle methylation in animals and plants, as animal mitochondrial tRNAs have m5C sites. Here we characterize 5 members of the RNA 5-methylcytosine family in Arabidopsis and extend the functional characterization of TRDMT1 and NOP2A/OLI2. We demonstrate that nuclear tRNA methylation requires two evolutionarily conserved methyltransferases, TRDMT1 and TRM4B. trdmt1 trm4b double mutants are hypersensitive to the antibiotic hygromycin B, demonstrating the function of tRNA methylation in regulating translation. Additionally we demonstrate that nuclear large subunit 25S rRNA methylation requires the conserved RNA methyltransferase NSUN5. Our results also suggest functional redundancy of at least two of the NOP2 paralogs in Arabidopsis.ConclusionsOur data demonstrates widespread occurrence and conservation of non-coding RNA methylation in the kingdom Plantae, suggesting important and highly conserved roles of this post-transcriptional modification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0580-8) contains supplementary material, which is available to authorized users.

RNA 5-Methylcytosine Analysis by Bisulfite Sequencing.

Cells have developed molecular machineries, which can chemically modify DNA and RNA nucleosides. One particular and chemically simple modification, (cytosine-5) methylation (m(5)C), has been detected both in RNA and DNA suggesting universal use of m(5)C for the function of these nucleotide polymers. m(5)C can be reproducibly mapped to abundant noncoding RNAs (transfer RNA, tRNA and ribosomal RNA, rRNA), and recently, also nonabundant RNAs (including mRNAs) have been reported to carry this modification. Quantification of m(5)C content in total RNA preparations indicates that a limited number of RNAs carry this modification and suggests specific functions for (cytosine-5) RNA methylation. What exactly is the biological function of m(5)C in RNA? Before attempting to address this question, m(5)C needs to be mapped specifically and reproducibly, preferably on a transcriptome-wide scale. To facilitate the detection of m(5)C in its sequence context, RNA bisulfite sequencing (RNA-BisSeq) has been developed. This method relies on the efficient chemical deamination of nonmethylated cytosine, which can be read out as single nucleotide polymorphism (nonmethylated cytosine as thymine vs. methylated cytosine as cytosine), when differentially comparing cDNA libraries to reference sequences after DNA sequencing. Here, the basic protocol of RNA-BisSeq, its current applications and limitations are described.

Function and Detection of 5-Methylcytosine in eukaryotic RNA

Modified nucleosides play an important role in RNA function and have been identified in multiple RNA types, including tRNAs, rRNAs, mRNAs and small regulatory RNAs. Among these, 5-methylcytosine (m(5)C) has been detected in rRNAs and tRNAs, and early reports suggested its presence in mRNAs. Known and well studied as an epigenetic mark in DNA, the prevalence and function of m(5)C in RNA is either incompletely explored (i.e., in tRNA and rRNA) or virtually unknown (i.e., in mRNA and other noncoding RNA). Two eukaryotic methyltransferases have been demonstrated to place m(5)C in RNA; however, their substrate specificity and cellular functions are not completely understood. With the recent development of m(5)C detection in RNA by bisulfite sequencing, comprehensive analyses to determine its occurrence and biological roles are now feasible. In this article we review the occurrence, function and biochemical detection of m(5)C in eukaryotic RNA, and provide perspectives on the biological roles of this modification in the transcriptome.

5-methylcytosine in RNA: detection, enzymatic formation and biological functions

The nucleobase modification 5-methylcytosine (m5C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m5C-methylation were extensively studied during the last decades. However, the location, the mechanism of formation and the cellular function(s) of the same modified nucleobase in RNA still remain to be elucidated. The recent development of a bisulfite sequencing approach for efficient m5C localization in various RNA molecules puts ribo-m5C in a highly privileged position as one of the few RNA modifications whose detection is amenable to PCR-based amplification and sequencing methods. Additional progress in the field also includes the characterization of several specific RNA methyltransferase enzymes in various organisms, and the discovery of a new and unexpected link between DNA and RNA m5C-methylation. Numerous putative RNA:m5C-MTases have now been identified and are awaiting characterization, including the identification of their RNA substrates and their related cellular functions. In order to bring these recent exciting developments into perspective, this review provides an ordered overview of the detection methods for RNA methylation, of the biochemistry, enzymology and molecular biology of the corresponding modification enzymes, and discusses perspectives for the emerging biological functions of these enzymes.

Crystal Structure of the Escherichia coli 23S rRNA:m 5C Methyltransferase RlmI (YccW) Reveals Evolutionary Links between RNA Modification Enzymes

Methylation is the most common RNA modification in the three domains of life. Transfer of the methyl group from S-adenosyl- l-methionine (AdoMet) to specific atoms of RNA nucleotides is catalyzed by methyltransferase (MTase) enzymes. The rRNA MTase RlmI ( rRNA large subunit methyltransferase gene I; previously known as YccW) specifically modifies Escherichia coli 23S rRNA at nucleotide C1962 to form 5-methylcytosine. Here, we report the crystal structure of RlmI refined at 2 Å to a final R-factor of 0.194 ( R free = 0.242). The RlmI molecule comprises three domains: the N-terminal PUA domain; the central domain, which resembles a domain previously found in RNA:5-methyluridine MTases; and the C-terminal catalytic domain, which contains the AdoMet-binding site. The central and C-terminal domains are linked by a β-hairpin structure that has previously been observed in several MTases acting on nucleic acids or proteins. Based on bioinformatics analyses, we propose a model for the RlmI–AdoMet–RNA complex. Comparative structural analyses of RlmI and its homologs provide insight into the potential function of several structures that have been solved by structural genomics groups and furthermore indicate that the evolutionary paths of RNA and DNA 5-methyluridine and 5-methylcytosine MTases have been closely intertwined.