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.

通过运用蛋白质组学和核糖体组学方法对翻译的开放阅读框进行识别，已揭示出成千上万的先前未注释的细胞微蛋白，即少于150个氨基酸残基的肽链，以及与经典蛋白共同编码的替代蛋白（alt-proteins），其长度可超过150个氨基酸。大多数微蛋白和替代蛋白尚未被充分表征，部分原因在于它们的短长度限制了与已知蛋白结构域同源性的分析。微蛋白和替代蛋白的亚细胞定位在很大程度上仍然未知，但其功能可能具有重大影响。基于邻近性的生物素化提供了一种吸引人的方法，以定义活细胞和动物中细胞亚室的经典蛋白组成。在此，我们开发了一种高通量技术，通过 TurboID（MicroID）的邻近性生物素化全球映射未注释的微蛋白和替代蛋白到亚细胞定位。我们展示了150多种微蛋白和替代蛋白与亚核器官以及执行关键细胞功能的复合体相关联。其中一种新型微蛋白，alt-LAMA3，定位于核仁并参与前核糖体RNA的转录。为了展示其在体内的应用价值，我们应用MicroID于小鼠模型中，鉴定了一种由假基因翻译的保守核微蛋白，并为进一步利用该技术在动物中微蛋白的发现和表征奠定了基础。