Single nucleotide resolution mapping and quantification of pre-rRNA processing and modification dynamics

Ribosome biogenesis involves the synthesis of precursor ribosomal RNAs (pre-rRNAs) and their processing (cleavage) into mature rRNAs. Traditional techniques like northern blotting and metabolic labeling, while robust, have limited resolution and throughput. Here, we present NanoRibolyzer, a long-read nanopore sequencing-based method that enables ab initio characterization and quantification of rRNA precursors. By combining supervised and unsupervised mapping approaches, it identifies both known and novel rRNA species and precisely characterizes cleavage sites at single-nucleotide resolution. Additionally, a simplified cell fractionation procedure allows for spatial resolution of nuclear and cytoplasmic pre-rRNAs. Targeted knockdown experiments further quantify specific intermediate accumulations, defining condition-specific 'fingerprints' with potential biomarker value. Lastly, using a novel precursor-specific RNA modification analysis, we revealed that unprocessed 47S pre-rRNA is already marked by pseudouridines, suggesting that these modifications are deposited co-transcriptionally in human. With its unique mapping strategy, NanoRibolyzer advances our understanding of pre-rRNA processing providing high-resolution insights into ribosome biogenesis.