Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcomeovercame these hurdles through the development of “Ribo-FilterOut”, which is based on the separation of footprints from ribosome subunits by ultrafiltration, and “Ribo-Calibration”, which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method revealed the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.基于核糖体足迹深度测序的核糖体谱分析（ribosome profiling），现已成为阐明蛋白质合成调控机制的有力工具。然而当前方法存在显著局限：存在核糖体RNA（rRNA）污染，且缺乏适用于测量转录本上核糖体数量的方法。本研究通过开发基于超滤法分离核糖体亚基与足迹的「Ribo-FilterOut」技术，以及依赖化学计量学定义的mRNA-核糖体复合物外源spike-in的「Ribo-Calibration」方法，攻克了上述难题。联合使用这两种方法，可在全基因组范围内实现转录本上的核糖体数目、翻译起始速率，以及转录本降解前的整体翻译事件总数的定量估算。此外，本方法还揭示了热休克应激、衰老进程以及不同细胞类型中的核糖体分配模式。本研究改进的核糖体谱分析策略，可实现转录组水平上细胞翻译的动力学与化学计量学参数的定量测定。