Accurate detection of m6A RNA modifications in native RNA sequences [Yeast]
Ontology highlight
ABSTRACT: The field of epitranscriptomics has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here we show that using Oxford Nanopore Technologies, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Our results open new avenues to investigate the universe of RNA modifications with single nucleotide resolution, in individual RNA molecules.
Project description:The field of epitranscriptomics has experienced a major revolution in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here we show that using the platform offered by Oxford Nanopore Technologies, we can detect N6-methyladenosine (m6A) RNA modifications with high accuracy. Our results open new avenues to investigate the universe of RNA modifications with single nucleotide, single molecule resolution.
Project description:Epitranscriptomics modifications constitute a gene expression checkpoint in all living organism including plants. Considering the relevance of nitrogen nutrition and metabolism for the correct plant growth and development, it can be hypothesized that epitranscriptome changes must regulate every biological process in plants including nitrogen nutrition. In the present work, the epritranscriptomics changes in maritime pine roots caused by ammonium nutrition have been monitored through direct RNA sequencing using Oxford Nanopore Technology. The main transcriptome responses to ammonium nutrition affected to transcripts involved in nitrogen and carbon metabolisms, defense response, hormone synthesis and signaling, and translation. Additionally to a global detection of epitranscriptomics marks, the m6A deposition and its dynamics have been identified, which seems to be important regulators of translation when compared with the proteomic profiles of the same samples. In this sense, the obtained results suggest that protein translation is finely regulated through the epitranscriptomics marks maybe through changes in mRNA polyA length, transcript amount and ribosome protein composition. The multiomics results in the present study suggest that the epitranscriptome must modulate the responses to development and environmental changes, including ammonium nutrition, through buffering, filtering and focusing the final products of the gene expression.
Project description:The transcriptome profiles of the model plant Arabidopsis thaliana have been extensively studied and charcaterised under different developmental and physiological conditions. However, most of these “RNA-sequencing” datasets have been generated using the sequencing of reverse-transcribed cDNAs from mRNAs that have a relatively short read length. Here, we performed direct RNA sequencing using the latest Oxford Nanopore Technology (ONT) with unusual read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been under-estimated. The ONT direct RNA sequencing technology identified transcript isoforms at a vegetative (14 day old seedlings, stage 1.04) and a reproductive stage (stage 6.00-6) when 10% of the flowers had opened. In-house software called TrackCluster was used to determine alternative transcription initiation (ATI), possible alternative polyadenylation (APA), poly(A) length, alternative splicing (AS), and fusion transcripts. Tombo software was used to detect RNA base modifications. More than 38,500 novel transcript isoforms were identified, including six categories of fusion-transcripts which may result from differential RNA processing mechanisms. Fusion-transcripts are prone to mis-assembly by sequencing with short reads using next-generation-sequencing (NGS). These new transcript isoforms provide important additions to the annotated Arabidopsis genome. The power of ONT in detecting RNA modifications was demonstrated by characterisation of the modifications between mobile mRNAs and total mRNAs. The mobile mRNAs were enriched in m5C modifications, which is consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT direct RNA sequencing greatly enhances the identification of novel RNA transcript isoforms and RNA base modifications.
Project description:In recent years, the topic of RNA modifications, epitranscriptomics, has gained momentum in a variety of fields. It is now firmly established that modifications of RNA can affect splicing, RNA stability and turnover, and translation, which ultimately influence the pathophysiology of organisms. Although its importance has been appreciated, epitranscriptomics in the heart has not been investigated extensively. Among over 140 RNA modifications, adenosine-to-inosine (A-to-I) RNA editing is of particular interest in the heart as it is reported to be increased in patients of congenital heart defects and atherosclerosis. However, its function in cardiomyocytes, especially during development, has not been investigated.
2020-05-05 | GSE141345 | GEO
Project description:The small RNA universe of Capitella teleta
Project description:Recent years has witnessed rapid progress of the field epitranscriptomics. Functional interpretation of epitranscriptome relies on mapping technologies which determine the localization and stoichiometry of various RNA modifications. However, contradictory results are derived from different studies, questioning the biological impacts of certain RNA modifications. Here, we develop an approach for the generation of synthetic RNA library resembling the endogenous transcriptome but lacking modifications. Incorporating this modification-free RNA library as a negative control into established techniques, we obtain precise and quantitative maps of m6A and m5C after removing the pervasive false positives resulted from other elements such as specific sequence context and RNA secondary structure.
Project description:We report the direct RNA sequencing of HEK293 and a primary human mammary epithelial cell (HMEC) line using Oxford Nanopore based sequencing. Using this data, we built an algorithm to detect m6A modifications within the DRACH motif context. Evaluation of m6A sites was carried out with HEK METTL3 knockdown and HMEC ALKBH5 over expression cell lines.
Project description:Separation and relative quantification by targeted mass spectrometry of the TNFRSC members pior and after treatment with a generic deubiquitinating enzyme