Redefining the human pre-rRNA 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.

核糖体生物发生通路（ribosomal biogenesis pathway）涵盖功能性核糖体的成熟与组装过程，对核糖体的正常形成至关重要。传统技术如Northern印迹法（northern blotting）已对主要的核糖体前体RNA（pre-rRNA）加工中间体进行了表征。然而，鉴于核糖体组装的复杂性，包括异常切割产物在内的大量pre-rRNA物种及副产物仍未被阐明。
本研究介绍了NanoRibolyzer——一款利用直接cDNA或RNA长读长纳米孔测序（Nanopore-seq）数据来检测并定量rRNA中间体的生物信息学流程。结合简化的细胞核分离流程，我们能够对细胞核与细胞质中的rRNA进行空间表征，区分天然多腺苷酸化与非多腺苷酸化的rRNA物种。
借助该工具，我们系统性地捕获了涵盖已知与新型中间体在内的大量pre-rRNA物种，实现了单核苷酸分辨率的已知及新鉴定加工位点的定位。在核糖体加工因子的靶向敲低实验中，我们有效定量了预期中间体的积累，并鉴定出条件特异性的加工“特征指纹”。
此外，我们还对pre-rRNA及成熟rRNA群体开展了前体特异性的RNA修饰分析，揭示了它们在核糖体生物发生通路中的复杂关联。总体而言，该工具为研究rRNA加工与修饰动态提供了前沿方法，为解析人类核糖体生物发生通路的复杂性提供了全新视角。