Transcription and translation are intertwined processes in which mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have left gaps in our understanding of critical biological processes. To address these gaps, we developed an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP) that capitalizes on advancements in long-read sequencing and RNA-base editing–mediated technologies to simultaneously profile translation and transcription at both the gene and mRNA isoform levels. We also developed the EditsC metric to quantify editing and leverage the single-molecule, full-length transcript information provided by long-read sequencing. Here, we report concordance between gene-level translation profiles obtained with long-read and short-read Ribo-STAMP. We show that LR-Ribo-STAMP successfully profiles translation of mRNA isoforms and links regulatory features, such as upstream open reading frames (uORFs), to translation measurements. We apply LR-Ribo-STAMP to discovering translational differences at both the gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia and find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions. We also discover regulatory elements that distinguish translational differences at the isoform level. We highlight GRK6, in which hypoxia is observed to increase expression and translation of a shorter mRNA isoform, giving rise to a truncated protein without the AGC Kinase domain. Overall, LR-Ribo-STAMP is an important advance in our repertoire of methods that measures mRNA translation with isoform sensitivity.

中文翻译：

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长读长 Ribo-STAMP 以亚型分辨率同时测量转录和翻译


转录和翻译是交织在一起的过程，其中 mRNA 亚型是关键的中介。然而，在 mRNA 亚型水平分析翻译的方法学局限性使我们对关键生物过程的理解存在空白。为了解决这些差距，我们开发了一个称为长读长 Ribo-STAMP （LR-Ribo-STAMP） 的集成计算和实验框架，该框架利用长读长测序和 RNA 碱基编辑介导的技术进步，同时在基因和 mRNA 亚型水平上分析翻译和转录。我们还开发了 EditsC 指标来量化编辑并利用长读长测序提供的单分子全长转录本信息。在这里，我们报告了通过长读长和短读 Ribo-STAMP 获得的基因水平翻译谱之间的一致性。我们表明 LR-Ribo-STAMP 成功地分析了 mRNA 亚型的翻译，并将调节特征（如上游开放阅读框 （uORF））与翻译测量联系起来。我们将 LR-Ribo-STAMP 应用于发现常氧和缺氧下三阴性乳腺癌细胞系中基因和亚型水平的翻译差异，发现 LR-Ribo-STAMP 有效地描绘了条件之间的正交转录和翻译转移。我们还发现了在亚型水平上区分翻译差异的调节元件。我们重点介绍了 GRK6，其中观察到缺氧增加了较短 mRNA 亚型的表达和翻译，从而产生没有 AGC 激酶结构域的截短蛋白。总体而言，LR-Ribo-STAMP 是我们以亚型敏感性测量 mRNA 翻译的方法库中的重要进步。