scholarly journals Accurate detection of m6A RNA modifications in native RNA sequences

2019 ◽  
Author(s):  
Huanle Liu ◽  
Oguzhan Begik ◽  
Morghan C Lucas ◽  
Christopher E. Mason ◽  
Schraga Schwartz ◽  
...  
Keyword(s):  
Rna Modifications ◽  
Rna Sequences ◽  
Single Nucleotide ◽  
Rna Molecules ◽  
Base Calling ◽  
Oxford Nanopore ◽  
Nucleotide Resolution ◽  
The Universe ◽  
Oxford Nanopore Technologies ◽  
Single Nucleotide Resolution

ABSTRACTThe 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.

scholarly journals Nucleotide resolution profiling of m3C RNA modification by HAC-seq

2020 ◽  
Author(s):  
Jia Cui ◽  
Qi Liu ◽  
Erdem Sendinc ◽  
Yang Shi ◽  
Richard I Gregory
Keyword(s):  
Rna Modification ◽  
Rna Modifications ◽  
Mcf7 Cells ◽  
Single Nucleotide ◽  
Novel Method ◽  
Specific Mapping ◽  
Nucleotide Resolution ◽  
And Function ◽  
Trna Isoacceptors ◽  
Single Nucleotide Resolution

Abstract Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called ‘epitranscriptome’, contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.

scholarly journals Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’

Open Biology ◽  
2017 ◽  
Vol 7 (5) ◽  
pp. 170077 ◽  
Author(s):  
Matthias Schaefer ◽  
Utkarsh Kapoor ◽  
Michael F. Jantsch
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Molecular Biology ◽  
Genetic Information ◽  
Biological Function ◽  
Rna Modifications ◽  
Single Nucleotide ◽  
Technological Advances ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the ‘epitranscriptome’. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications.

AlkAniline‐Seq: Profiling of m 7 G and m 3 C RNA Modifications at Single Nucleotide Resolution

2018 ◽  
Vol 57 (51) ◽  
pp. 16785-16790 ◽  
Author(s):  
Virginie Marchand ◽  
Lilia Ayadi ◽  
Felix G. M. Ernst ◽  
Jasmin Hertler ◽  
Valérie Bourguignon‐Igel ◽  
...  
Keyword(s):  
Rna Modifications ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals TIF-Seq2 disentangles overlapping isoforms in complex human transcriptomes

2020 ◽  
Vol 48 (18) ◽  
pp. e104-e104 ◽  
Author(s):  
Jingwen Wang ◽  
Bingnan Li ◽  
Sueli Marques ◽  
Lars M Steinmetz ◽  
Wu Wei ◽  
...  
Keyword(s):  
Human Cell Line ◽  
Rna Seq ◽  
Single Nucleotide ◽  
Rna Molecules ◽  
Unannotated Transcript ◽  
Long Read ◽  
Nucleotide Resolution ◽  
Overlapping Transcripts ◽  
Single Nucleotide Resolution ◽  
Leukaemia Patient

Abstract Eukaryotic transcriptomes are complex, involving thousands of overlapping transcripts. The interleaved nature of the transcriptomes limits our ability to identify regulatory regions, and in some cases can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptomes, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5′ and 3′ ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identified thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-Seq, we accurately defined boundaries of lowly expressed unannotated and read-through transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explored transcription regulation among overlapping units and investigated their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription units. Our work shows that, by paring the 5′ and 3′ end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitate accurate assignment of promoters to genes and easily identify transcriptionally fused genes.

AlkAniline‐Seq: Profiling of m 7 G and m 3 C RNA Modifications at Single Nucleotide Resolution

2018 ◽  
Vol 130 (51) ◽  
pp. 17027-17032
Author(s):  
Virginie Marchand ◽  
Lilia Ayadi ◽  
Felix G. M. Ernst ◽  
Jasmin Hertler ◽  
Valérie Bourguignon‐Igel ◽  
...  
Keyword(s):  
Rna Modifications ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

2020 ◽  
Author(s):  
Justin T. Roberts ◽  
Allison M. Porman ◽  
Aaron M. Johnson
Keyword(s):  
Chemical Properties ◽  
Rna Modifications ◽  
Single Nucleotide ◽  
Uv Crosslinking ◽  
Rna Immunoprecipitation ◽  
Input Sample ◽  
Library Complexity ◽  
Rna Fragments ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractMethylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m6A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

Sign in / Sign up

Export Citation Format

Share Document