Comprehensive identification of diverse ribosomal RNA modifications by targeted nanopore direct RNA sequencing and JACUSA2.
Isabel S Naarmann-de VriesChristiane ZorbasAmina LemsaraMichael PiechottaFelix G M ErnstLudivine WacheulDenis L J LafontaineChristoph DieterichPublished in: RNA biology (2023)
Ribosomal RNAs are decorated by numerous post-transcriptional modifications whose exact roles in ribosome biogenesis, function, and human pathophysiology remain largely unknown. Here, we report a targeted direct rRNA sequencing approach involving a substrate selection step and demonstrate its suitability to identify differential modification sites in combination with the JACUSA2 software. We compared JACUSA2 to other tools designed for RNA modification detection and show that JACUSA2 outperforms other software with regard to detection of base modifications such as methylation, acetylation and aminocarboxypropylation. To illustrate its widespread usability, we applied our method to a collection of CRISPR-Cas9 engineered colon carcinoma cells lacking specific enzymatic activities responsible for particular rRNA modifications and systematically compared them to isogenic wild-type RNAs. Besides the numerous 2'-O methylated riboses and pseudouridylated residues, our approach was suitable to reliably identify differential base methylation and acetylation events. Importantly, our method does not require any prior knowledge of modification sites or the need to train complex models. We further report for the first time detection of human rRNA modifications by direct RNA-sequencing on Flongle flow cells, the smallest-scale nanopore flow cell available to date. The use of these smaller flow cells reduces RNA input requirements, making our workflow suitable for the analysis of samples with limited availability and clinical work.
Keyphrases
- single cell
- induced apoptosis
- endothelial cells
- crispr cas
- cell cycle arrest
- loop mediated isothermal amplification
- wild type
- real time pcr
- dna methylation
- induced pluripotent stem cells
- healthcare
- cancer therapy
- genome editing
- genome wide
- electronic health record
- cell death
- endoplasmic reticulum stress
- gene expression
- transcription factor
- cell therapy
- oxidative stress
- density functional theory
- quantum dots
- histone deacetylase
- mass spectrometry
- health information
- cell proliferation
- sensitive detection
- hydrogen peroxide
- pi k akt
- heat shock
- molecular dynamics
- heat shock protein