RNA molecules contain more than just the four standard nucleotide bases. After RNA is produced, cells often modify individual nucleotides through a variety of chemical changes that can influence RNA stability, structure, and function. One of the most common of these modifications is pseudouridine.

Pseudouridine is the most abundant post-transcriptional modification found in human ribosomal RNA (rRNA), the RNA component of ribosomes that helps cells manufacture proteins. More than 110 pseudouridine sites have been identified in human rRNA, and the amount of modification at each site can vary depending on cell type, tissue, or biological condition.

Accurately measuring pseudouridine levels across the transcriptome has traditionally been challenging. Existing methods often require labor-intensive workflows or are limited to analyzing predefined sets of modification sites. As interest in epitranscriptomics continues to grow, researchers are seeking scalable approaches capable of profiling RNA modifications at high resolution.

Researchers have now demonstrated that nanopore direct RNA sequencing can accurately identify and quantify pseudouridine modifications across human ribosomal RNA. The work was led by researchers at the University of Sherbrooke in Quebec, Canada.

Unlike conventional RNA sequencing methods that require conversion of RNA into complementary DNA, nanopore direct RNA sequencing analyzes native RNA molecules directly. This allows chemical modifications present on the RNA molecule to influence the electrical signals generated during sequencing, creating an opportunity to detect modified nucleotides.

The research team evaluated the performance of the Dorado v5.1 nanopore sequencing model using samples from human liver tissue, induced pluripotent stem cells, and HeLa cells. Results were compared against pseudouridine sites that had previously been validated using mass spectrometry.

The analysis showed that nanopore sequencing successfully detected 95 of 117 validated pseudouridine sites. Even more importantly, the approach accurately quantified modification stoichiometry, the percentage of RNA molecules carrying a modification at a specific location, for approximately 85% of the validated sites.

The measurements were highly reproducible across experiments, demonstrating that nanopore sequencing can generate consistent results for epitranscriptomic studies.

The researchers also investigated factors that limited detection performance. They found that regions with low GC content were the primary source of missed modification sites, suggesting an area where future improvements in sequencing models may further increase accuracy.

These findings are significant because they demonstrate that nanopore direct RNA sequencing can move beyond simple sequence determination and provide quantitative information about RNA modifications at individual nucleotide positions.

As researchers continue exploring the role of RNA modifications in development, disease, and cellular regulation, scalable technologies for modification profiling will become increasingly important. The ability to combine RNA sequencing and epitranscriptomic analysis in a single workflow could help accelerate discoveries in fields ranging from cancer biology to stem cell research.

By providing reproducible, site-specific measurements of pseudouridine abundance, nanopore sequencing offers a promising platform for studying one of the most widespread RNA modifications in human cells.