RESUMO
PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.
Assuntos
Prolina , Ligação Proteica , Domínios WW , Humanos , Motivos de Aminoácidos , Modelos Moleculares , Prolina/metabolismo , Prolina/química , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Espalhamento a Baixo Ângulo , Spliceossomos/metabolismo , Difração de Raios XRESUMO
The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.
Assuntos
Receptores de Antígenos de Linfócitos T , Timócitos , Animais , Camundongos , Diferenciação Celular/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Timócitos/metabolismo , Timo/metabolismoRESUMO
Serine protease inhibitor clade E member 1 (SERPINE1) inhibits extracellular matrix proteolysis and cell detachment. However, SERPINE1 expression also promotes tumor progression and plays a crucial role in metastasis. Here, we solve this apparent paradox and report that Serpine1 mRNA per se, independent of its protein-coding function, confers mesenchymal properties to the cell, promoting migration, invasiveness, and resistance to anoikis and increasing glycolytic activity by sequestering miRNAs. Expression of Serpine1 mRNA upregulates the expression of the TRA2B splicing factor without affecting its mRNA levels. Through transcriptional profiling, we found that Serpine1 mRNA expression downregulates through TRA2B the expression of genes involved in the immune response. Analysis of human colon tumor samples showed an inverse correlation between SERPINE1 mRNA expression and CD8+ T cell infiltration, unveiling the potential value of SERPINE1 mRNA as a promising therapeutic target for colon tumors.
RESUMO
Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA-protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events ( https://github.com/malewins/Plant-iCLIPseq ), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites ( https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html ).
Assuntos
Nucleotídeos , RNA , Animais , RNA/genética , Nucleotídeos/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Ribonucleases/metabolismo , Imunoprecipitação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mamíferos/genéticaRESUMO
Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
Assuntos
RNA , Ubiquitina-Proteína Ligases , Humanos , RNA/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Formaldeído/toxicidade , Aldeídos/toxicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.
Assuntos
Sítios de Splice de RNA , Splicing de RNA , Humanos , Sítios de Splice de RNA/genética , Íntrons/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of â¼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.
Assuntos
Perfil de Ribossomos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biossíntese de Proteínas , Perfil de Ribossomos/métodos , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.
Assuntos
Mecanismo Genético de Compensação de Dose , Cromossomo X , Masculino , Feminino , Animais , Camundongos , Metilação , Cromossomo X/genética , Mamíferos/genética , Estabilidade de RNARESUMO
Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.
Assuntos
Antígenos CD19 , Ribonucleoproteínas Nucleares Heterogêneas , Leucemia de Células B , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas de Ligação a RNA , Humanos , Sítios de Ligação , Epitopos , Ribonucleoproteínas Nucleares Heterogêneas/genética , Mutação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas de Ligação a RNA/genética , Leucemia de Células B/genéticaRESUMO
The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.
Assuntos
Proteínas de Ligação a RNA , Fatores de Transcrição , Humanos , Proteínas de Ligação a DNA/genética , Epilepsia , Corpos de Processamento , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Sítios de Splice de RNA , Processamento Alternativo/genética , Antígenos CD19/genética , Antígenos CD19/metabolismo , Epitopos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Mutagênese/genética , Mutação , Recidiva Local de Neoplasia/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Isoformas de Proteínas/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.
Assuntos
Proteínas de Ciclo Celular , Metiltransferases , Adenosina/análogos & derivados , Animais , Proteínas de Ciclo Celular/metabolismo , Metilação , Metiltransferases/genética , Camundongos , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.
Assuntos
Adenosina/análogos & derivados , Aprendizado de Máquina , Processamento Pós-Transcricional do RNA , RNA-Seq/métodos , Adenosina/química , Adenosina/metabolismo , Animais , Células HEK293 , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Motivos de Nucleotídeos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA-Seq/normas , Sensibilidade e EspecificidadeRESUMO
Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While "exitrons" are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed "falsitrons," that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.
Assuntos
Processamento Alternativo , Artefatos , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Transcrição Reversa , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Éxons , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoterapia/métodos , Íntrons , Modelos Biológicos , Conformação de Ácido Nucleico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , RNA Mensageiro/química , RNA Mensageiro/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.
Assuntos
Adenosina/análogos & derivados , Axônios/fisiologia , Proteínas de Drosophila/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neurônios/citologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína do X Frágil da Deficiência Intelectual/genética , Neurônios/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genéticaRESUMO
In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.
Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Fosfoproteínas/metabolismo , RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Éxons , Técnicas de Silenciamento de Genes , Humanos , Proteínas com Domínio LIM/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Fatores de Processamento de Serina-Arginina/química , Fatores de Processamento de Serina-Arginina/genética , Fatores de Transcrição/genética , Transcriptoma , TransfecçãoRESUMO
Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of â¼50% were detected in defined amplicons, and this threshold can be lowered to â¼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.