A Versatile Method for Cell-Specific Profiling of Translated mRNAs in Drosophila

In Drosophila melanogaster few methods exist to perform rapid cell-type or tissue-specific expression profiling. A translating ribosome affinity purification (TRAP) method to profile actively translated mRNAs has been developed for use in a number of multicellular organisms although it has only been implemented to examine limited sets of cell- or tissue-types in these organisms. We have adapted the TRAP method for use in the versatile GAL4/UAS system of Drosophila allowing profiling of almost any tissue/cell-type with a single genetic cross. We created transgenic strains expressing a GFP-tagged ribosomal protein, RpL10A, under the control of the UAS promoter to perform cell-type specific translatome profiling. The GFP::RpL10A fusion protein incorporates efficiently into ribosomes and polysomes. Polysome affinity purification strongly enriches mRNAs from expected genes in the targeted tissues with sufficient sensitivity to analyze expression in small cell populations. This method can be used to determine the unique translatome profiles in different cell-types under varied physiological, pharmacological and pathological conditions.

在黑腹果蝇（Drosophila melanogaster）中，可用于快速开展细胞类型或组织特异性表达谱分析的方法十分有限。目前已有针对多种多细胞生物开发的翻译核糖体亲和纯化（translating ribosome affinity purification, TRAP）技术，可用于分析活跃翻译中的mRNA，但在这些生物中，该技术仅被应用于检测有限的细胞或组织类型集合。本研究将TRAP技术适配至黑腹果蝇通用的GAL4/UAS表达系统中，仅需一次遗传杂交即可实现几乎所有组织/细胞类型的翻译组谱分析。我们构建了在UAS启动子调控下表达绿色荧光蛋白（GFP）标记的核糖体蛋白RpL10A的转基因品系，以开展细胞类型特异性的翻译组谱分析。GFP::RpL10A融合蛋白可高效整合至核糖体与多聚核糖体中。多聚核糖体亲和纯化可从靶组织中高效富集目标基因的mRNA，且灵敏度足以分析少量细胞群体中的基因表达情况。该技术可用于解析不同细胞类型在不同生理、药理及病理条件下的特异性翻译组谱特征。