Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single base resolution mapping of 2'-O-methylation sites in human mRNA and in 3' terminal ends of small RNAs.

ABSTRACT: The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could identify 2'OMe sites at single base resolution. Nm-seq is particularly useful for identifying 2'OMe sites located at the 3' terminal ends of small RNAs. Here, we present an optimized protocol for Nm-seq and a protocol for applying Nm-seq to identify 2'OMe sites on small RNA 3' terminal ends.

SUBMITTER: Hsu PJ

PROVIDER: S-EPMC6397781 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Single base resolution mapping of 2'-O-methylation sites in human mRNA and in 3' terminal ends of small RNAs.

Hsu Phillip J PJ Fei Qili Q Dai Qing Q Shi Hailing H Dominissini Dan D Ma Lijia L He Chuan C

Methods (San Diego, Calif.) 20181122

The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could id ...[more]

PMID: 30471344

Similar Datasets

Nm-seq maps 2'-O-methylation sites in human mRNA with base precision.

Project description:The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.

| S-EPMC5712428 | biostudies-literature

Corrigendum: Nm-seq maps 2'-O-methylation sites in human mRNA with base precision.

Project description:[This corrects the article PMC5712428.].

| S-EPMC6994179 | biostudies-literature

Nm-Mut-seq: a base-resolution quantitative method for mapping transcriptome-wide 2'-O-methylation.

Project description: Not available

| S-EPMC10474006 | biostudies-literature

Identification of sites of 2'-O-methylation vulnerability in human ribosomal RNAs by systematic mapping.

Project description:Ribosomal RNA modifications are important in optimizing ribosome function. Sugar 2'-O-methylation performed by fibrillarin-associated box C/D antisense guide snoRNAs impacts all steps of translation, playing a role in disease etiology (cancer). As it renders adjacent phosphodiester bonds resistant to alkaline treatment, 2'-O-methylation can be monitored qualitatively and quantitatively by applying next-generation sequencing to fragments of randomly cleaved RNA. We remapped all sites of 2'-O-methylation in human rRNAs in two isogenic diploid cell lines, one producing and one not producing the antitumor protein p53. We identified sites naturally modified only partially (confirming the existence in cells of compositionally distinct ribosomes with potentially specialized functions) and sites whose 2'-O-methylation is sensitive to p53. We mapped sites particularly vulnerable to a reduced level of the methyltransferase fibrillarin. The remarkable fact that these are largely sites of natural hypomodification provides initial insights into the mechanism of partial RNA modification. Sites where methylation appeared vulnerable lie peripherally on the 3-D structure of the ribosomal subunits, whereas the numerous modifications present at the core of the subunits, where the functional centers lie, appeared robustly made. We suggest that vulnerable sites of 2'-O-methylation are highly likely to undergo specific regulation during normal and pathological processes.

| S-EPMC5597630 | biostudies-literature

High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis.

Project description:N(6)-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification.

| S-EPMC3956118 | biostudies-literature

How do base-pairing small RNAs evolve?

Project description:The increasing numbers of characterized base-pairing small RNAs (sRNAs) and the identification of these regulators in a broad range of bacteria are allowing comparisons between species and explorations of sRNA evolution. In this review, we describe some examples of trans-encoded base-pairing sRNAs that are species-specific and others that are more broadly distributed. We also describe examples of sRNA orthologs where different features are conserved. These examples provide the background for a discussion of mechanisms of sRNA evolution and selective pressures on the sRNAs and their mRNA target(s).

| S-EPMC4542690 | biostudies-literature

High-resolution RNA 3'-ends mapping of bacterial Rho-dependent transcripts.

Project description:Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3'-5' exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3' ends of hundreds of Escherichia coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3'-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3'-5' exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3'-ends of Rho-dependent transcripts in E. coli.

| S-EPMC6061677 | biostudies-literature

Selective terminal methylation of a tRNA wobble base.

Project description:Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.

| S-EPMC5909439 | biostudies-literature

Direct quantification of 3' terminal 2'-<i>O</i>-methylation of small RNAs by RT-qPCR.

Project description:Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2'-hydroxyl-group of ribonucleotides (2'-O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3' terminal 2'-O-methylation, it is difficult to monitor the dynamic change of 3' terminal 2'-O-methylation during various biological processes. Capitalizing on the finding that 3' terminal RNA 2'-O-methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2'-O-methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2'-O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2'-O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1).

| S-EPMC6191718 | biostudies-literature

Improved Placement of Multi-mapping Small RNAs.

Project description:High-throughput sequencing of small RNAs (sRNA-seq) is a popular method used to discover and annotate microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). One of the key steps in sRNA-seq data analysis is alignment to a reference genome. sRNA-seq libraries often have a high proportion of reads that align to multiple genomic locations, which makes determining their true origins difficult. Commonly used sRNA-seq alignment methods result in either very low precision (choosing an alignment at random), or sensitivity (ignoring multi-mapping reads). Here, we describe and test an sRNA-seq alignment strategy that uses local genomic context to guide decisions on proper placements of multi-mapped sRNA-seq reads. Tests using simulated sRNA-seq data demonstrated that this local-weighting method outperforms other alignment strategies using three different plant genomes. Experimental analyses with real sRNA-seq data also indicate superior performance of local-weighting methods for both plant miRNAs and heterochromatic siRNAs. The local-weighting methods we have developed are implemented as part of the sRNA-seq analysis program ShortStack, which is freely available under a general public license. Improved genome alignments of sRNA-seq data should increase the quality of downstream analyses and genome annotation efforts.

| S-EPMC4938663 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data