Redefining the human pre-rRNA nanopore Sequencing. Redefining the human pre-rRNA processing at single nucleotide resolution using long read Nanopore sequencing

The ribosomal biogenesis pathway, which includes the maturation and assembly of functional ribosomes, is crucial for proper formation of the ribosomes. Traditional techniques like northern blotting have characterized major pre-rRNA processing intermediates. However, considering the complexity of ribosome assembly, a wide range of pre-rRNA species and by-products, including aberrantly cleaved, remain uncharacterized. Here, we introduce NanoRibolyzer, a bioinformatic pipeline that uses direct cDNA or RNA long-read Nanopore-seq data to detect and quantify rRNA intermediates. Coupled with a simplified nuclei isolation procedure, we were able to spatially characterize nuclear and cytoplasmic rRNA, distinguishing naturally polyadenylated and non-polyadenylated species. Using our tool, we systematically captured a broad range of pre-rRNA species, including both known and novel intermediates, allowing us to map established and newly identified processing sites with single-nucleotide resolution. In targeted knockdown experiments of ribosome processing factors, we effectively quantified the accumulation of expected intermediates and identified condition-specific processing “fingerprints”. Lastly, we performed precursor-specific RNA modification analyses across pre- and mature rRNA populations, highlighting their intricate relationships within the ribosomal biogenesis pathway. Overall, such tool offers a state-of-the-art approach to studying rRNA processing and modification dynamics, providing novel insights into the complexities of the human ribosome biogenesis pathway.