Ghatpande Vighnesh, Paul Uma, Howard MacKenzie A, Cenik Can
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA.
Departments of Neurology and Neuroscience, Center for Learning and Memory, Dell Medical School, University of Texas at Austin, Austin, Texas 78712, USA.
bioRxiv. 2025 Aug 4:2025.08.04.668486. doi: 10.1101/2025.08.04.668486.
In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this challenge using Ribo-ITP on difficult-to-collect samples like microdissected hippocampal tissues and single pre-implantation embryos. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed distinct sample specific expression patterns of the detected translons. To test the translational capacity of the identified translons, we engineered a translon-dependent GFP reporter system and detected expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.
在过去十年中，通过核糖体图谱分析和蛋白质组学发现了数量出乎意料的大量翻译区域（翻译子）。翻译子可调节mRNA翻译并编码微肽，这些微肽有助于多蛋白复合物的形成、肌肉中的钙调节以及胚胎发育过程中的信号传导。然而，由于传统核糖体图谱分析和质谱分析对样本输入量要求较高，翻译子的鉴定一直局限于细胞系或大型器官。在此，我们使用核糖体免疫沉淀测序技术（Ribo-ITP）来应对这一挑战，该技术适用于如显微切割的海马组织和单个植入前胚胎等难以收集的样本。对广泛细胞类型中的一千多个核糖体图谱数据集进行的比较分析揭示了所检测到的翻译子具有明显的样本特异性表达模式。为了测试已鉴定翻译子的翻译能力，我们构建了一个依赖于翻译子的绿色荧光蛋白（GFP）报告系统，并在小鼠胚胎干细胞（mESCs）中检测了在近同源起始密码子处起始的翻译子的表达。对mESCs中的翻译子进行突变，发现一小部分翻译子对细胞生长有负面影响。综上所述，我们开展了一项概念验证研究，以从低输入量样本中鉴定非经典翻译事件，该方法可应用于传统方法难以处理的细胞和组织类型。
