Mapping subcellular localizations of unannotated microproteins with MicroID

Identification of translated small open reading frames using proteogenomic approaches and ribosomal profiling has revealed thousands of previously unannotated cellular microproteins, or polypeptides of less than 150 amino acids, and alternative proteins (alt-proteins) that are co-encoded with canonical proteins and can be longer than 150 amino acids. The majority of microproteins and alt-proteins remain uncharacterized, in part because their short lengths preclude analysis of homology to known protein domains. The subcellular localizations of microproteins and alt-proteins remain largely unknown but can have significant implications for their functions. Proximity-dependent biotinylation has provided an attractive approach to define the canonical protein composition of subcellular compartments in living cells and animals. Here, we developed a high-throughput technology for global mapping of unannotated microproteins and alt-proteins to subcellular localizations by proximity-dependent biotinylation with TurboID (MicroID). We showed that more than 150 microproteins and alt-proteins are associated with subnuclear organelles and with complexes that carry out critical cellular functions. One of these novel microproteins, alt-LAMA3, localizes to the nucleolus and functions in pre-rRNA transcription. As a demonstration of its in vivo utility, we applied MicroID in a mouse model, identifying a conserved nuclear microprotein translated from a pseudogene, and establishing the foundation for use of this technology in discovery and characterization of microproteins in animals.

利用蛋白质基因组学（proteogenomics）方法与核糖体谱分析（ribosomal profiling）对翻译后的小开放阅读框进行鉴定，已发现数千种此前未被注释的细胞微蛋白（microproteins，即长度不足150个氨基酸的多肽），以及与经典蛋白（canonical proteins）共编码、长度可超过150个氨基酸的替代蛋白（alt-proteins）。绝大多数微蛋白与替代蛋白仍未得到功能表征，部分原因在于其较短的长度阻碍了针对已知蛋白质结构域（protein domains）的同源性分析。微蛋白与替代蛋白的亚细胞定位（subcellular localizations）在很大程度上仍不明确，但该定位信息对其功能具有重要意义。邻近依赖型生物素标记法（proximity-dependent biotinylation）为定义活细胞与活动物体内亚细胞区室（subcellular compartments）的经典蛋白组成提供了一种极具吸引力的研究手段。本研究开发了一种高通量技术，通过采用TurboID（MicroID）的邻近依赖型生物素标记法，实现对未注释微蛋白与替代蛋白的亚细胞定位全局图谱绘制。研究表明，超过150种微蛋白与替代蛋白与核内亚细胞器（subnuclear organelles）以及执行关键细胞功能的复合物相关联。其中一种新型微蛋白alt-LAMA3定位于核仁（nucleolus），并在前核糖体RNA（pre-rRNA）转录过程中发挥功能。为验证该技术的体内（in vivo）应用价值，我们在小鼠模型中应用了MicroID，鉴定出一种由假基因（pseudogene）翻译而来的保守核微蛋白，为该技术在动物体内的微蛋白发现与表征研究奠定了基础。