Unveiling Tissue-Specific RNA Landscapes in Mouse Organs During Fasting and Feeding Using Nanopore Direct RNA Sequencing

Understanding tissue-specific RNA landscapes is essential for uncovering the functional mechanisms of key organs in mammals. However, current knowledge remains limited, as short-read RNA sequencing—the predominant method for assessing gene expression—depends on incomplete gene annotations and struggles to resolve the diverse transcripts produced by genes. To address these limitations, we used an integrative approach combining nanopore direct RNA sequencing (DRS), ATAC-Seq, and short-read RNA-seq. This method enabled the analysis of RNA landscapes across major mouse organs under fasting and fed conditions, representing two extremes of the caloric cycle. Our study uncovered tens of thousands of novel transcripts and identified hundreds of genes with tissue-specific expression, revealing additional layers of regulated pathways within each organ that conventional short-read RNA-seq cannot resolve. By profiling transcript expression across multiple organs under identical conditions, we conducted comparative analyses exposing significant differences in transcript isoforms and regulations. Moreover, nanopore DRS revealed dynamic changes in poly(A) tail length and m6A modifications of transcripts, many regulated in a tissue-specific manner. These changes likely contribute to functional differentiation and metabolic specialization of various organs. Collectively, our findings reveal previously unrecognized layers of gene regulation, offering new insights into the metabolic basis of organ function.

解析组织特异性RNA表达谱，对于揭示哺乳动物关键器官的功能机制至关重要。然而，当前相关认知仍较为有限：作为评估基因表达的主流技术，短读长RNA测序依赖于不完整的基因注释，且难以解析基因所产生的多种转录本。为解决上述局限，我们采用了整合纳米孔直接RNA测序（nanopore direct RNA sequencing, DRS）、ATAC测序（ATAC-Seq）与短读长RNA测序的一体化分析策略。该策略可实现小鼠主要器官在禁食与饱腹状态下的RNA表达谱分析——这两种状态分别代表了能量循环的两个极端。本研究鉴定出数以万计的全新转录本，并发现了数百个具有组织特异性表达的基因，揭示了传统短读长RNA测序无法解析的各器官内调控通路的新增层级。通过在统一实验条件下对多器官的转录本表达进行谱型分析，我们开展了比较研究，揭示了转录本异构体与调控模式间的显著差异。此外，纳米孔DRS揭示了转录本的poly(A)尾长度与m6A甲基化修饰的动态变化，其中多数变化呈现组织特异性调控特征。上述变化或可推动不同器官的功能分化与代谢特化。综上，本研究的发现揭示了此前未被认知的基因调控层级，为理解器官功能的代谢基础提供了全新见解。