K562 RNA modification detection using IVT coupled with nanopore sequencing

RNA modifications are critical for transcript function and regulation. Direct RNA nanopore sequencing offers a unique advantage by observing these modifications through characteristic alterations in ionic current signals. Various computational tools have been developed to detect RNA modifications, typically by comparing a sample of interest with a control lacking the target modification, often achieved through enzyme knockdown or knockout. We have developed a robust in vitro transcription protocol to generate a modification-free copy of any input RNA to detect all modifications that alter the raw ionic current signal relative to the canonical nucleotides during nanopore sequencing. We generated an in vitro transcribed sample from K562 cells and used Nanocompore to detect 26,619 modified RNA sites across 2,520 transcript isoforms from 1,742 genes. Of these, 56% are consistent with the well-characterized m6A modification. By inferring the identities of these modifications, we assessed differential usage and correlation patterns, revealing a significant co-occurrence between m6A and m5C modifications within the same transcripts. Additionally, some modifications were non-randomly associated with alternative splicing events. This study provides a comprehensive survey of RNA modifications across the transcriptome, demonstrating the utility of in vitro transcription coupled with direct RNA nanopore sequencing to simultaneously detect multiple modifications without the need for additional independent biochemical assays. The protocol is consistent in the future with more complex experimental designs, for example for differential modification usage between samples.

RNA修饰对于转录本的功能与调控至关重要。直接RNA纳米孔测序（direct RNA nanopore sequencing）凭借其可通过离子电流信号的特征性变化直接观测RNA修饰的特性，具备独特优势。目前已有多款计算工具被开发用于RNA修饰检测，此类工具通常通过对比待测样本与缺失目标修饰的对照样本实现检测，而对照样本的制备常通过酶敲低（enzyme knockdown）或酶敲除（enzyme knockout）完成。本研究建立了一套稳定可靠的体外转录（in vitro transcription）实验流程，可针对任意输入RNA生成无修饰的转录本拷贝，从而在纳米孔测序过程中，识别所有相较于经典核苷酸（canonical nucleotides）会改变原始离子电流信号的RNA修饰位点。我们从K562细胞中制备了一份体外转录样本，并借助Nanocompore工具，在来自1742个基因的2520个转录本异构体中检测到26619个RNA修饰位点。其中56%的位点与已被充分表征的N6-甲基腺苷（m6A）修饰一致。通过推断这些修饰的类型，我们评估了修饰的差异使用模式与关联特征，发现同一转录本内的m6A与5-甲基胞嘧啶（m5C）修饰之间存在显著的共现现象。此外，部分RNA修饰与可变剪接（alternative splicing）事件呈现非随机的关联关系。本研究对转录组（transcriptome）范围内的RNA修饰进行了全面调研，证实了将体外转录与直接RNA纳米孔测序相结合的实验方案，可在无需额外独立生化实验的前提下同时检测多种RNA修饰。该实验流程未来可适配更复杂的实验设计，例如用于比较不同样本间的修饰差异使用情况。