Salussolia Catherine L, Winden Kellen D, Sahin Mustafa
F.M. Kirby Neurobiology Center, Rosamund Stone Zander Translational Neuroscience Center, Department of Neurology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Bio Protoc. 2022 May 5;12(9):e4407. doi: 10.21769/BioProtoc.4407.
Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as well as between cell populations, contributing to the diversity of mammalian tissues. Earlier methods that analyzed gene expression at the level of translation, such as polysomal- or ribosomal-profiling, required large amounts of starting material to isolate enough RNA for analysis by microarray or RNA-sequencing. Thus, rare or less abundant cell types within tissues were not able to be properly studied with these methods. Translating ribosome affinity purification (TRAP) utilizes the incorporation of an eGFP-affinity tag on the large ribosome subunit, driven by expression of cell-type specific Cre-lox promoters, to allow for identification and capture of transcripts from actively translating ribosomes in a cell-specific manner. As a result, TRAP offers a unique opportunity to evaluate the entire mRNA translation profile within a specific cell type, and increase our understanding regarding the cellular complexity of mammalian tissues. Schematic demonstrating TRAP protocol for identifying ribosome-bound transcripts specifically within cerebellar Purkinje cells.
哺乳动物组织高度异质且复杂，这给理解调控各种组织内蛋白质表达的分子机制带来了挑战。最近的研究表明，核糖体水平的翻译受到高度调控，并且可以独立于转录组水平观察到的基因表达以及细胞群体之间的表达而变化，这导致了哺乳动物组织的多样性。早期在翻译水平分析基因表达的方法，如多核糖体或核糖体分析，需要大量起始材料来分离足够的RNA用于微阵列或RNA测序分析。因此，这些方法无法对组织中罕见或丰度较低的细胞类型进行适当研究。翻译核糖体亲和纯化（TRAP）利用细胞类型特异性Cre-lox启动子的表达驱动，在大核糖体亚基上掺入eGFP亲和标签，从而以细胞特异性方式鉴定和捕获来自活跃翻译核糖体的转录本。因此，TRAP为评估特定细胞类型内的整个mRNA翻译谱提供了独特的机会，并增进了我们对哺乳动物组织细胞复杂性的理解。图为展示TRAP方案的示意图，该方案用于在小脑浦肯野细胞中特异性鉴定核糖体结合的转录本。
