m6A In RNA Research Articles

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Thymopentin promotes ovarian angiogenesis in mice by activating N6-methyladenosine (m6A) RNA modification of key factors in the Notch/Tie1 pathway

Abstract: BACKGROUND: Premature ovarian failure (POF) is a typical condition of pathological ovarian ageing. Injuries and atrophies of blood vessels in the ovaries can lead to ovarian insufficiency, but the underlying mechanism remains unclear. AIMS: This study investigated the epigenetic mechanism by which thymopentin (TP-5) activated ovarian angiogenesis to achieve its effects on POF. MATERIALS AND METHODS: The qPCR, western blot and immunofluorescence staining were used to detecte the expression levels of gene. The hematoxylin and eosin stainingwas used to histopathological examination. The RNA-Seq sequencing was used to transcriptome detection. RESULTS: First, pathological examination revealed that TP-5 significantly increased ovary weight in the cyclophosphamide-induced POF mouse model. The proportion of atretic follicles was reduced, and the level of E2 in the peripheral blood was elevated. Meanwhile, immunofluorescence staining showed that TP-5 increased protein expression of CD31 and Tie1 in the ovarian tissues of mice with POF, suggesting that TP-5 could induce ovarian angiogenesis. RNA-Seq sequencing results suggested that TP-5 could induce the high level of Notch/Tie2 pathway expression that is associated with angiogenesis in ovarian tissues. qPCR and western blotting showed significantly increased expression of METTL3, a methyltransferase that catalyses the formation of N6-methyladenosine (m6A) in RNA. Meanwhile, the results of the dot blot suggested that the overall RNA methylation level was significantly higher in the ovarian tissues of mice in the TP-5 group than in the control group. Finally, RIP-PCR analysis showed that specific sites in the 3'-untranslated region (3'UTR) of the mRNA of key factors in the Notch/Tie2 pathway were hypermethylated in the ovaries of mice in the TP-5 treatment group. CONCLUSION: Therefore, this study demonstrated that TP-5 exerted its therapeutic effect on POF by stimulating angiogenesis. The mechanism of action is the promotion of the expression of RNA m6A methyltransferases by TP5, which increases the overall RNA m6A methylation level in mouse ovarian tissue. Specifically, the methylation ofspecific m6A sites in the 3'UTR of the RNA of key factors in the Notch/Tie2 pathway increased, which enhanced their stability and expression levels, leading to the induction of angiogenesis.

Profiling of N6-Methyladenosine in Zika Virus RNA and Host Cellular mRNA.

N6-Methylation of adenosine (m6A) in viral RNA was first identified in Rous sarcoma virus, influenza virus, and SV40 virus several decades ago. More recently, human immunodeficiency virus-1, hepatitis C virus, and Zika virus (ZIKV) RNAs have also been reported to be modified by m6A, highlighting the significance of this posttranscriptional RNA modification. Nevertheless, the functions of m6A and their roles in the context of viral infections are not completely understood. Similarly, the mechanisms by which viral infections may alter the dynamics of the host RNA methylome have yet to be elucidated. Here, we describe an experimental protocol to obtain m6A profiles of ZIKV RNA and the host cell mRNA using methylated RNA immunoprecipitation-sequencing (MeRIP-Seq).

Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the+1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.

Abstract 584: FTO-Dependent m6A Regulates Cardiomyocyte and Cardiac Function During Remodeling and Repair

Background: Despite its functional importance in various fundamental bioprocesses, the studies of N6-methyladenosine (m6A) in the heart are lacking. In this study, we investigated the role of fat mass and obesity-associated (FTO), an m6A demethylase, in cardiac contractile function during homeostasis and remodeling. Methods: We used clinical human samples, preclinical pig and mouse models and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO using AAV9 (in vivo), adenovirus (both in vivo and in vitro) and siRNAs (in vitro). We performed methylated (m6A) RNA immunoprecipitation sequencing (MeRIP-seq) to map transcriptome-wide m6A, and MeRIP qPCR assays to map and validate m6A in individual transcripts, in healthy and failing hearts and myocytes. We performed proteomics analysis using stable isotope labeling with amino acids in cell culture to study m6A role in mRNA to protein translation. Results: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is carried out by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. Conclusion: Collectively, our study demonstrates the functional importance of FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

N6-Methylation Assessment in Escherichia coli 23S rRNA Utilizing a Bulge Loop in an RNA-DNA Hybrid.

We propose a sequence-selective assay of N6-methyl-adenosine (m6A) in RNA without PCR or reverse transcription, by employing a hybridization assay with a DNA probe designed to form a bulge loop at the position of a target modified nucleotide. The m6A in the bulge in the RNA-DNA hybrid was assumed to be sufficiently mobile to be selectively recognized by an anti-m6A antibody with a high affinity. By employing a surface-plasmon-resonance measurement or using a microtiter-plate immunoassay method, a specific m6A in the Escherichia coli 23S rRNA sequence could be detected at the nanomolar level when synthesized and purified oligo-RNA fragments were used for measurement. We have successfully achieved the first selective detection of m6A2030 specifically in 23S rRNA from real samples of E. coli total RNA by using our immunochemical approach.

N6-Methyladenosine modification of lincRNA 1281 is critically required for mESC differentiation potential.

Previous studies have revealed the critical roles of N6-methyladenosine (m6A) modification of mRNA in embryonic stem cells (ESCs), but the biological function of m6A in large intergenic noncoding RNA (lincRNA) is unknown. Here, we showed that the internal m6A modification of linc1281 mediates a competing endogenous RNA (ceRNA) model to regulate mouse ESC (mESC) differentiation. We demonstrated that loss of linc1281 compromises mESC differentiation and that m6A is highly enriched within linc1281 transcripts. Linc1281 with RRACU m6A sequence motifs, but not an m6A-deficient mutant, restored the phenotype in linc1281-depleted mESCs. Mechanistic analyses revealed that linc1281 ensures mESC identity by sequestering pluripotency-related let-7 family microRNAs (miRNAs), and this RNA-RNA interaction is m6A dependent. Collectively, these findings elucidated the functional roles of linc1281 and its m6A modification in mESCs and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns.

Role of DNA and RNA N6-Adenine Methylation in Regulating Stem Cell Fate.

Epigenetic modifications have been evidenced to participate in eukaryotic stem cell fate decision. Among the most studied, 5-methylcytosine (m5C) and its derivatives are wellestablished epigenetic codes that play important roles in stem cell pluripotency and differentiation. Based on improved detection techniques, recent studies have succeeded in defining N6-adenine methylation (m6A) in eukaryotic DNA and RNA. The abundant m6A methylation in RNA was shown to be involved in multiple cellular metabolisms while the presence and functional potential of DNA m6A methylation in different species advanced our knowledge in the m6A-mediated biological processes. m6A modification has been observed during embryogenesis and has been proposed to fine-tune stem cell regulation. The m6A methyltransferases and demethylases work together to control the dynamic state of m6A marks in genomic DNA and RNA to ensure proper cell fate transition and determination, which are vital to the development and survival of eukaryotes.

N6-Allyladenosine: A New Small Molecule for RNA Labeling Identified by Mutation Assay.

RNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N6-allyladenosine (a6A) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. a6A behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells. We also show that human RNA N6-methyladenosine (m6A) methyltransferases METTL3/METTL14 can work with a synthetic cofactor, namely allyl-SAM (S-adenosyl methionine with methyl replaced by allyl) in order to site-specifically install an allyl group to the N6-position of A within specific sequence to generate a6A-labeled RNAs. The iodination of N6-allyl group of a6A under mild buffer conditions spontaneously induces the formation of N1,N6-cyclized adenosine and creates mutations at its opposite site during complementary DNA synthesis of reverse transcription. The existing m6A in RNA is inert to methyltransferase-assisted allyl labeling, which offers a chance to differentiate m6A from A at individual RNA sites. Our work demonstrates a new method for RNA labeling, which could find applications in developing sequencing methods for nascent RNAs and RNA modifications.

RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response (DDR), detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 minutes) and transiently induced at DNA damage sites in response to UV. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL31 and the demethylase FTO2. In the absence of METTL3 catalytic activity, cells showed delayed repair of UV-induced cyclobutane pyrimidine (CPD) adducts and elevated sensitivity to UV, demonstrating the importance of m6A in the UV-responsive DDR. Multiple DNA polymerases are involved in the UV response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair (NER) pathway3, while others participate in trans-lesion synthesis (TLS) to allow replication past damaged lesions in S phase4. DNA polymerase κ (Pol κ), which has been implicated in both NER and TLS5,6, required the catalytic activity of METTL3 for immediate localization to UV-induced DNA damage sites. Importantly, Pol κ over-expression qualitatively suppressed the CPD removal defect associated with METTL3 loss. Taken together, we have uncovered a novel function for RNA m6A modification in the UV-induced DDR, and our findings collectively support a model whereby m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Dynamics of Human and Viral RNA Methylation during Zika Virus Infection.

Infection with the flavivirus Zika (ZIKV) causes neurological, immunological, and developmental defects through incompletely understood mechanisms. We report that ZIKV infection affects viral and human RNAs by altering the topology and function of N6-adenosine methylation (m6A), a modification affecting RNA structure and function. m6A nucleosides are abundant in ZIKV RNA, with twelve m6A peaks identified across full-length ZIKV RNA. m6A in ZIKV RNA is controlled by host methyltransferases METTL3 and METTL14 and demethylases ALKBH5 and FTO, and knockdown of methyltransferases increases, while silencing demethylases decreases, ZIKV production. YTHDF family proteins, which regulate the stability of m6A-modified RNA, bind to ZIKV RNA, and their silencing increases ZIKV replication. Profiling of the m6A methylome of host mRNAs reveals that ZIKV infection alters m6A location in mRNAs, methylation motifs, and target genes modified by methyltransferases. Our results identify a mechanism by which ZIKV interacts with and alters host cell functions.

M6A: A novel hallmark of translation

The discovery of N6-methyladenosine (m6A) in polyadenylated RNA can be traced back to the 1970s. However, the biological functions of m6A in mammalian mRNA had remained mysterious for decades. This...

N 6-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens

N6-Methyladenine (m6A) is the most abundant internal modification on mammalian mRNA. Very recently, m6A has been reported as a potentially important 'epigenetic' mark in eukaryotes. Until now, site-specific detection of m6A is technically very challenging. Here, we first reveal that m6A significantly hinders DNA- and RNA-directed DNA synthesis. Systematic investigations of 5'-triphosphates of a variety of 5-substituted 2'-deoxyuridine analogs in primer extension have been performed. In the current study, a quantitative analysis of m6A in the RNA or DNA context has been achieved, using Bst DNA polymerase catalyzed primer extension. Molecular dynamics study predicted that m6A in template tends to enter into and be restrained in the MGR region of Bst DNA polymerase, reducing conformational flexibility of the DNA backbone. More importantly, a site-specific determination of m6A in human ribosomal RNA (rRNA) with high accuracy has been afforded. Through a cumulative analysis of methylation alterations, we first reveal that significantly cancer-related changes in human rRNA methylation were present in patients with hepatocellular carcinoma.

Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m(5)C), 2'-O-methylcytidine (Cm), N(6)-methyladenosine (m(6)A), and 2'-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m(5)C, Cm and Am are tissue-specific. In addition, the 2'-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m(5)C and m(6)A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m(5)C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m(6)A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.

The beginning of RNA.

In October 1993, Reinhard Luhrmann organized a small RNA meeting in Marburg, Germany, and invited me as a speaker. This was the same year the Nobel Prize for Splicing was awarded, and many of the meeting talks concerned the mechanism of splicing. I was excited that Joan Steitz, who had first proposed that snRNPs were involved in splicing, was the Keynote Speaker at this conference. She had also recently been named the President-Elect of the newly formed RNA Society. Two interacting low abundance snRNPs, U11 and U12, had been discovered the year before in the Steitz lab, but their function was unknown. My lab had recently discovered that the U11 snRNA binds to a Rous sarcoma virus (RSV) RNA element that helps regulate the balance between spliced and unspliced viral RNA. This was before AT-AC introns were discovered, and U11 and U12 snRNPs were found to substitute for U1 and U2 snRNPs in a minor class of spliceosome. I flew to Frankfurt from Baltimore, found the train station and then had to figure out how to buy a train ticket from a non-English speaking ticket machine. Finally, I arrived in Marburg, a bit jet-lagged, and went to the meeting hotel. Tim Nilsen, whom I barely knew, was sitting in the lobby when I checked in. He immediately attacked me for a critical review that he thought I had written six years earlier of his paper on m6A methylation of influenza RNA. It is possible I did write the offending review, but his paper was published nevertheless. That was when I learned that Tim never forgets a thing. I thought the meeting had a great mix of people working on mRNA splicing and on viral RNAs. Other invited speakers from the US included Bob Krug and Mike Malim. Bob had made the amazing discovery that influenza virus stole caps from cellular mRNAs. Mike studied Rev-mediated export of unspliced HIV RNA. Incidentally, Bob had also identified m6A in the influenza genomic RNA in the '70s, when my group had begun to study it in RSV. Tim talked about the role of U6 snRNP in cis and trans splicing in nematodes. Reinhard talked about his lab's detailed analysis of snRNPs and of the spliceosome. The meeting was very stimulating and interactive, ending with a dinner and dance party at Reinhard's home. Marburg turned out to be a charming small German town with a castle, despite its infamy as the site of an outbreak of Marburg virus (a relative of Ebola). I remember walking around town a lot. On one of these walks back to the hotel, Joan shared her frustration with Tim and me that the journal Cell did not appreciate RNA work. This conversation led to the idea that a new journal should be started that focused on RNA. By the end of the Marburg meeting, Tim had agreed to start such a journal and to be Editor in Chief. He asked me to be the Editor for Viral RNA. I don't think I believed he would pull it off, so I didn't leap at the chance. I had no idea it would become the leading journal for the RNA community and regret that I didn't accept his offer. To my surprise, the first copy of RNA appeared less than a year and a half later, in March 1995, as the journal for the RNA Society. It has always been both rigorous and fun to read. It has an important role as a place to get fast and fair reviews of papers on any aspect of RNA biology. I recommend it to both my graduate students and colleagues as a reasonable place to send a paper that will be seen by the RNA community. The timing of its beginning was propitious, as there has been an explosion of new findings in the RNA field in the last 20 years. I have been fascinated with the discoveries of new types of RNAs, including microRNAs and long noncoding RNAs. Genome sequencing revealed there were fewer protein-coding genes than expected, requiring massive alternative splicing to synthesize mRNAs for all the missing proteins. Mechanisms of nonsense-mediated mRNA decay (NMD) remain interesting and controversial. Finally, the m6A field seems to be undergoing a rebirth with many functions ascribed to cellular mRNAs containing this internal modification that was originally studied in viral RNAs decades ago. It is hard to imagine the RNA field without the RNA journal.

Structures of human m6A RNA demethylases: FTO and ALKBH5

Methylation at the N6 position of adenosine in mRNA (m6A) was first observed around 40 years ago but little was known about the function of this modification at the time of discovery. Research over the last ten years has led to the identification of methyltransferases that produce or `write' the m6A RNA mark and recently demethylases that remove or `erase' m6A marks, thus indicating that the m6A modification is reversible. Recent analyses have identified the motif and regions where m6A marks are present within RNA transcripts as well as m6A binding protein `readers'. These studies have revealed a role for m6A methylation in mRNA stability and demonstrated that sequence specific subsets of transcripts can be regulated in this manner. Like the histone lysine JmjC demethylases, nucleic acid demethylases are members of the 2-oxoglutarate and iron dependent oxygenase superfamily. The human nucleic acid oxygenases (NAOXs) with known structures include the TET enzyme that produces 5-hydroxymethylcytosine in DNA, ALKBH2 and 3 that demethylate 3-methylcytosine and 1-methyladenine in DNA, and ALKBH8 that modifies tRNA. We identified the fat mass and obesity associated protein, FTO, as a NAOX1and m6A in RNA was recently reported to be a bona-fide substrate of FTO in in-vivo. Subsequently, ALKBH5 was also shown to be an m6A demethylase. Here we present structural studies of FTO2and ALKBH53as well as biochemical work that has led to structurally informed inhibitor design of the m6A demethylases for the development of small molecules to probe biological function and for potential use as therapeutics.

Mechanism and Function of Oxidative Reversal of DNA and RNA Methylation

The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/α-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N(6)-methyladenosine (m(6)A) in RNA, indicating that members of this subfamily of dioxygenases have a general function in demethylating nucleic acids. In this review, we discuss recent advances in this emerging field, focusing on the mechanism and function of TET-mediated DNA demethylation.

N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.

We report here that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to an increased level of m6A in mRNA, whereas overexpression of FTO resulted in a decreased level of m6A in human cells. We further show that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.

Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations.

We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA of Rous sarcoma virus (RSV). The results of that study and of experiments using inhibitors of methylation suggest that m6A might be involved in mRNA processing events. We describe an approach for directly analyzing the function of m6A in RNA and for studying the sequence specificity of the m6A methylase. Two sites of methylation in RSV (nucleotides 7414 and 7424) were altered by oligonucleotide-directed mutagenesis. The highly conserved GAC consensus sequence at those sites was changed to GAU. The new sequences were no longer methylated in the RSV genomic RNA; the GAC sequence was required for efficient base modification at those two adenosines. The altered m6A pattern did not affect viral RNA processing or the viral life cycle within infected cells.

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.