Identification and perturbation of pseudouridine modifications in human mRNAs DOI Open Access
Sepideh Tavakoli

Published: Jan. 1, 2023

Pseudouridine (Ψ) is one of the most prevalent modifications in RNA, with its presence detected across various RNA types, including mRNA. The association Ψ different diseases, particularly cancers, and critical role tRNA structure underscores biological significance. However, robust detection this modification remains a formidable challenge, specific function within mRNA sequences unknown. In initial segment thesis, semi-quantitative method developed that leverages direct long-read nanopore sequencing for identifying pseudouridylated sites on human mRNAs. This approach capitalizes U-to-C basecalling error observed at positions. A comparative analysis involving modification-free transcriptome reveals depth coverage k-mer are pivotal parameters accurately detecting pseudouridine modifications.By fine-tuning these to account errors, our successfully identifies well-established pseudouridylation previously unreported uridine-modified sites. Many newly identified align k-mers targeted by synthases. Our workflow enables native molecules from data, showcasing capability discern multiple same strand. Expanding investigation, we introduce concepts type I II hypermodifications. Type hypermodifications denote instances high occupancy (>40%) single position, providing insights into extent On other hand, signify scenarios where more than occurs transcript. nuanced categorization adds understanding pseudouridine's influence, paving way comprehensive exploration processes. second part explore dynamics as cellular state. We use cultured SH-SY5Y neuroblastoma cells model system investigate impact cell state changes differentiating neuron-like assess alterations pseudouridine-modified patterns upon exposure lead, an environmental neurotoxin implicated developmental disorders. assign positions their cognate synthase performing siRNA knockdown experiments PUS7 TRUB1 validating targets using algorithm, Mod-p ID. find many fall coding sequences. also modified three categories: 1. Modification level all states (static), 2. levels differ (plastic), 3. depends change (condition-dependent plastic). Interestingly, motif tend be static, while can either static or plastic depending These studies provide exciting candidates future individual modifications. --Author's abstract

Language: Английский

Advances in nanopore direct RNA sequencing DOI Creative Commons
Miten Jain, Robin Abu-Shumays, Hugh E. Olsen

et al.

Nature Methods, Journal Year: 2022, Volume and Issue: 19(10), P. 1160 - 1164

Published: Oct. 1, 2022

Nanopore direct RNA sequencing (DRS) reads continuous native strands. Early adopters have used this technology to document nucleotide modifications and 3′ polyadenosine tails on strands without added chemistry steps. Individual ranging in length from 70 26,000 nucleotides been sequenced. In our opinion, broader acceptance of nanopore DRS by molecular biologists cell will be accelerated higher basecall accuracy lower input requirements.

Language: Английский

Citations

123
Enhanced detection of RNA modifications and read mapping with high-accuracy nanopore RNA basecalling models DOI
Gregor Diensthuber, Leszek P. Pryszcz, Laia Llovera

et al.

Genome Research, Journal Year: 2024, Volume and Issue: 34(11), P. 1865 - 1877

Published: Sept. 13, 2024

In recent years, nanopore direct RNA sequencing (DRS) became a valuable tool for studying the epitranscriptome, owing to its ability detect multiple modifications within same full-length native molecules. Although can be identified in form of systematic basecalling "errors" DRS data sets,

Language: Английский

Citations

10
Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling DOI Creative Commons
Albert Perlas, Tim Reska, Guillaume Croville

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 2, 2024

Abstract Avian influenza virus (AIV) currently causes a panzootic with extensive mortality in wild birds, poultry, and mammals, thus posing major threat to global health underscoring the need for efficient monitoring of its distribution evolution. Here, we utilized well-defined AIV strain systematically investigate characterization through rapid, portable nanopore sequencing by (i) benchmarking performance fully RNA extraction viral detection; (ii) comparing latest DNA approaches in-depth profiling; (iii) evaluating various computational pipelines consensus sequence creation phylogenetic analysis. Our results show that RNA-specific nanopores can accurately genomically profile from native while additionally detecting epigenetic modifications. We further identified an optimal laboratory bioinformatic pipeline reconstructing genomes data at rarefaction thresholds, which validated application real-world environmental samples livestock. Author Summary tested portable, easy-to-use technology obtain more information about potentially zoonotic avian virus, or AIV. has spread globally via migratory paths endangers domestic human populations given past evidence infections different animal species. here used novel genomic is based on explore virus; established optimized ways creating genome either directly converted DNA. then applied protocol dust were collected duck farm France during outbreak. showed able use resulting reconstruct relationship between responsible outbreak previously detected Altogether, how support surveillance pathogens recreating better understand evolution transmission these pathogens.

Language: Английский

Citations

5
Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications DOI Creative Commons
Lu Tan, Zhihao Guo, Xiaoming Wang

et al.

mSystems, Journal Year: 2024, Volume and Issue: 9(2)

Published: Jan. 31, 2024

Modifications on viral RNAs (vRNAs), either genomic or RNA transcripts, have complex effects the life cycle and cellular responses to infection. The advent of Oxford Nanopore Technologies Direct Sequencing provides a new strategy for studying modifications. To this end, multiple computational tools been developed, but systemic evaluation their performance in mapping vRNA modifications is lacking. Here, 10 were tested using Sindbis virus (SINV) isolated from infected mammalian (BHK-21) mosquito (C6/36) cells, with

Language: Английский

Citations

4
YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity DOI Creative Commons
Emily M. King,

Amanda Midkiff,

Karsyn McClain

et al.

Journal of Virology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 4, 2025

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), progressive neurodegenerative disease. Regulation of viral gene expression plays key role in persistence pathogenesis. However, the molecular mechanisms underlying this fine-tuned regulation remain poorly understood. Little known regarding RNA chemical modifications HTLV-1 how these affect biology disease development. Post-transcriptional modification common eukaryotes, with N 6 -methyladenosine (m A) being most prevalent. In study, we investigated m A on expression. Using MeRIP-Seq, mapped sites to 3’ end genome. We found RNA, as well oncogene transcripts tax hbz , contained modifications. A-depletion HTLV-1-transformed cells decreased sense-derived genes ( Tax, Gag, Env ) increased antisense-derived Hbz Tax were bound by reader proteins YTHDF1 YTHDC1 panel lines. vectors shRNA-mediated knockdown, that had opposing effects expression, decreasing increasing . Upon further abundance dependent deposition. The nuclear protein affected both sense- specifically enhanced export transcript. Collectively, our results demonstrate global levels regulate IMPORTANCE pathogenesis are controlled through tight fate can be epigenetic impact without altering DNA sequence. Our study details N6-methyladenosine reductions other genes, whereas suggest oncogenic transcripts, A-modified cells. interpreted YTHDC1, which dictate RNA. Understanding offers potential insights into novel therapeutic strategies diseases.

Language: Английский

Citations

0
Challenges to mapping and defining m6A function in viral RNA DOI Open Access
Stacy M. Horner, Matthew G. Thompson

RNA, Journal Year: 2024, Volume and Issue: 30(5), P. 482 - 490

Published: March 26, 2024

Viral RNA molecules contain multiple layers of regulatory information. This includes features beyond the primary sequence, such as structures and modifications, including N6-methyladenosine (m 6 A). Many recent studies have identified presence location m A in viral found diverse roles for this modification during infection. However, to date, mapping strategies limitations that prevent a complete understanding function on individual molecules. While sites been profiled bulk from many viruses, resulting maps RNAs described date present composite picture across infected cell. Thus, most it is unknown if unique profiles exist throughout infection, nor they regulate specific life cycle stages. Here, we describe several challenges defining provide framework future help how regulates

Language: Английский

Citations

3
Three-dimensional DNA nanoamplifiers actuated by demethylase-activated deoxyribozyme for the ultrasensitive detection of FTO in human breast tissues DOI
Liping Fan,

Xing-Miao Wang,

Juan Li

et al.

Sensors and Actuators B Chemical, Journal Year: 2025, Volume and Issue: unknown, P. 137431 - 137431

Published: Feb. 1, 2025

Language: Английский

Citations

0
Genomics-driven approaches for identifying viral virulence factors and developing antiviral therapies DOI
Rajiv Kumar Mondal,

Biraj Sarkar,

Amit Ghosh

et al.

Elsevier eBooks, Journal Year: 2025, Volume and Issue: unknown, P. 317 - 343

Published: Jan. 1, 2025

Language: Английский

Citations

0
Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS DOI Creative Commons
Alice Baek, Asif Rayhan, Ga-Eun Lee

et al.

Methods and Protocols, Journal Year: 2024, Volume and Issue: 7(1), P. 7 - 7

Published: Jan. 10, 2024

The biological significance of chemical modifications to the ribonucleic acid (RNA) human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding site-specific and context-dependent roles these remains limited, primarily due absence nucleotide-resolution mapping modification sites. In this study, we present a method for achieving sites on HIV-1 RNA using liquid chromatography tandem mass spectrometry (LC–MS/MS). LC–MS/MS, powerful tool capable directly analyzing native RNAs, proven effective in small molecules, including ribosomal transfer RNA. longer RNAs have posed challenges, such as 9 Kb virion RNA, complexity ambiguity differences among RNase T1-cleaved fragments LC-MS/MS data. Here, introduce new target enrichment isolate local that potentially harbor N6-methyladenosine (m6A) modifications. initial trial, used target-specific DNA probes only encountered insufficient fragmentation inefficient S1 digestion near site. Recognizing by is likely formation secondary structures proximity site, designed multiple annealing various better control substrates digestion. use non-target significantly improved isolation more homogeneous approximately 50 bases length. Oligonucleotide analysis isolated successfully separated detected both m6A-methylated non-methylated oligomers at two m6A-predicted principle strategy holds promise should be broadly applicable any lengthy was previously deemed infeasible investigation oligonucleotide LC-MS/MS.

Language: Английский

Citations

2
Current progress in strategies to profile transcriptomic m6A modifications DOI Creative Commons

Yuening Yang,

Yanming Lu, Yan Wang

et al.

Frontiers in Cell and Developmental Biology, Journal Year: 2024, Volume and Issue: 12

Published: July 11, 2024

Various methods have been developed so far for detecting N 6-methyladenosine (m6A). The total m6A level or the status at individual positions on mRNA can be detected and quantified through some sequencing-independent biochemical methods, such as LC/MS, SCARLET, SELECT, m6A-ELISA. However, m6A-detection techniques relying high-throughput sequencing more effectively advanced understanding about biological significance of m6A-containing pathway a transcriptomic over past decade. SGS-based (Second Generation Sequencing-based) with different detection principles widely employed this purpose. These include m6A-enrichment using antibodies, discrimination from unmodified A-base by nucleases, fusion protein strategy RNA-editing enzymes, marking chemical/biochemical reactions. Recently, TGS-based (Third brought new trend direct m6A-detection. This review first gives brief introduction current knowledge biogenesis function, then comprehensively describes m6A-profiling strategies including their principles, procedures, features. will guide users to pick appropriate according research goals, give insights developing novel in varying areas, continue expand our boundary m6A.

Language: Английский

Citations

2