Nanopore Sequencing Enables High-Throughput Quantification of Pseudouridine Modifications in Human Ribosomal RNA
Researchers benchmarked nanopore direct RNA sequencing against mass spectrometry-validated data and found it could detect 95 of 117 known pseudouridine sites in human ribosomal RNA with high reproducibility. Pseudouridine is the most common post-transcriptional RNA modification, with over 110 annotated sites in human ribosomal RNA, but existing methods for studying it are either low-throughput or limited to predefined panels. The findings suggest nanopore sequencing could serve as a scalable, site-resolved tool for studying how RNA modifications vary across cell types and biological conditions.
A study posted to bioRxiv evaluated nanopore direct RNA sequencing — using the Dorado v5.1 model — as a method for detecting and quantifying pseudouridine modifications in human ribosomal RNA. The researchers benchmarked the approach against mass spectrometry-validated pseudouridine sites across three biological systems: human liver tissue, induced pluripotent stem cells, and HeLa cells. Nanopore sequencing successfully detected 95 of 117 validated sites and accurately quantified modification stoichiometry at approximately 85% of those sites, with strong reproducibility across samples. The primary limitation identified was reduced performance in low GC-content sequence environments, which accounted for most detection failures. Current alternatives for pseudouridine profiling are either low-throughput or restricted to pre-selected site panels, making scalability a significant bottleneck in epitranscriptomic research. The authors conclude that nanopore sequencing represents a viable, high-throughput platform for comprehensive pseudouridine profiling across diverse human cell types and tissues.
What's missing
The study does not address whether the Dorado v5.1 model's performance generalizes to pseudouridine sites outside ribosomal RNA, such as in messenger RNA or other non-coding RNAs. It is also unclear whether the 22 undetected sites represent a systematic biological blind spot or are recoverable with model improvements. As a preprint, these findings have not yet undergone peer review.
What different sources said
- bioRxivCenter
Nanopore Direct RNA Sequencing Enables Reproducible, Site-Resolved Pseudouridine Quantification in Human Ribosomal RNA
Related
Gut Bacteria Enzyme Found to Break Down Heat-Processed Food Compounds, Producing Novel Biogenic Amines
Researchers have discovered that an enzyme in common gut bacteria can degrade N-epsilon-carboxymethyllysine (CML), a compound formed during thermal food processing, producing previously unknown biogenic amines. The enzyme, ornithine decarboxylase SpeC from enterobacteria, acts on CML and related modified lysine derivatives through a low-level 'underground' catalytic activity. This finding suggests a previously unrecognized communication axis between thermally processed dietary compounds and gut microbial physiology, with potential implications for host health.
Full-Length Gene Sequencing Reveals Two Distinct Bacterial Communities in Black-Legged Ticks Expanding Into Canada
Researchers used Oxford Nanopore full-length 16S rRNA gene sequencing to characterize the microbiome of Ixodes scapularis black-legged ticks collected in Nova Scotia, Canada, distinguishing between tick-adapted bacteria and environmentally acquired bacteria. The study comes as I. scapularis — the primary vector of Lyme disease — is rapidly expanding northward into Canada due to climate change. The findings suggest that environmentally derived bacteria in tick microbiomes are not mere contamination, which has implications for how tick microbiome data is collected and interpreted across surveillance studies.
Study Identifies Metabolic Link Between Cell Envelope Stress and Biofilm Formation in Bacteria
Researchers have discovered that the metabolite acetyl-CoA directly inhibits enzymes that degrade the bacterial signaling molecule c-di-GMP, connecting cell envelope biosynthesis stress to biofilm formation in Pseudomonas aeruginosa. The study found that sub-inhibitory concentrations of antibiotics targeting early peptidoglycan biosynthesis — but not other antibiotic classes — elevate c-di-GMP levels by reducing phosphodiesterase activity, with acetyl-CoA competing for the enzyme active site. Because the relevant enzyme domain is broadly conserved across bacterial species, this checkpoint mechanism may be widespread and could have implications for understanding antibiotic-induced biofilm responses.