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 The RNA Atlas, a single nucleotide resolution map of the human transcriptome

2019 ◽  
Author(s):  
Lucia Lorenzi ◽  
Hua-Sheng Chiu ◽  
Francisco Avila Cobos ◽  
Stephen Gross ◽  
Pieter-Jan Volders ◽  
...  
Keyword(s):  
Single Nucleotide ◽  
Human Transcriptome ◽  
Abundance Estimates ◽  
Rna Biology ◽  
Functional Regulation ◽  
Non Coding Rnas ◽  
Nucleotide Resolution ◽  
And Function ◽  
Polyadenylated Rnas ◽  
Single Nucleotide Resolution

AbstractThe human transcriptome consists of various RNA biotypes including multiple types of non-coding RNAs (ncRNAs). Current ncRNA compendia remain incomplete partially because they are almost exclusively derived from the interrogation of small- and polyadenylated RNAs. Here, we present a more comprehensive atlas of the human transcriptome that is derived from matching polyA-, total-, and small-RNA profiles of a heterogeneous collection of nearly 300 human tissues and cell lines. We report on thousands of novel RNA species across all major RNA biotypes, including a hitherto poorly-cataloged class of non-polyadenylated single-exon long non-coding RNAs. In addition, we exploit intron abundance estimates from total RNA-sequencing to test and verify functional regulation by novel non-coding RNAs. Our study represents a substantial expansion of the current catalogue of human ncRNAs and their regulatory interactions. All data, analyses, and results are available in the R2 web portal and serve as a basis to further explore RNA biology and function.

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 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 Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

RNA ◽  
2013 ◽  
Vol 19 (12) ◽  
pp. 1848-1856 ◽  
Author(s):  
N. Liu ◽  
M. Parisien ◽  
Q. Dai ◽  
G. Zheng ◽  
C. He ◽  
...  
Keyword(s):  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Rna Modification ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

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