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.

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 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.

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 Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma

2016 ◽  
Vol 90 (23) ◽  
pp. 10811-10822 ◽  
Author(s):  
Kübra Altinel ◽  
Kosuke Hashimoto ◽  
Yu Wei ◽  
Christine Neuveut ◽  
Ishita Gupta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Hepatitis B Virus ◽  
Human Liver ◽  
Single Nucleotide ◽  
B Virus ◽  
Human Livers ◽  
Nucleotide Resolution ◽  
Cap Analysis ◽  
Single Nucleotide Resolution

ABSTRACTHepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5′ ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers.IMPORTANCEDespite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5′ ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.

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 Deciphering Poxvirus Gene Expression by RNA Sequencing and Ribosome Profiling

2015 ◽  
Vol 89 (13) ◽  
pp. 6874-6886 ◽  
Author(s):  
Zhilong Yang ◽  
Shuai Cao ◽  
Craig A. Martens ◽  
Stephen F. Porcella ◽  
Zhi Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vaccinia Virus ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Single Nucleotide ◽  
Late Genes ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution ◽  
Reading Frames

ABSTRACTThe more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome.IMPORTANCEThis report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological advances allowing examination of the translated regions of mRNAs including start sites at single-nucleotide resolution. Vaccinia virus ribosome-associated mRNA sequences were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h after infection. The ribosome profiling approach was particularly valuable for poxviruses because of the close spacing of approximately 200 open reading frames and extensive transcriptional read-through resulting in overlapping mRNAs. The expression of intermediate and late genes, in particular, was visualized with unprecedented clarity and quantitation. We also identified novel putative translation initiation sites that were mostly associated with short protein coding sequences. The results provide a framework for further studies of poxvirus gene expression.

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.

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