In many organisms, tissue complexity and cellular diversity create a barrier that can hinder our understanding of gene expression programs. To address this problem, methods have been developed that allow for easy isolation of translated mRNAs from genetically defined cell populations. A prominent example is the Translating Ribosome Affinity Purification method also called TRAP. Here, ribosome associated mRNAs are isolated via purification of the ribosomal protein RPL10A/uL1, which is expressed under the control of a tissue specific promoter. Originally developed to investigate gene expression in mouse neurons, it has by now been adopted to many different organisms and tissues. Interestingly, TRAP has never been used successfully to analyze mRNA translation in germ cells. Employing a combination of genetic and biochemical approaches, I assessed several ribosomal proteins for their suitability for TRAP using the Caenorhabditis elegans germline as a target tissue. Surprisingly, I found that RPL10A/uL1 is not the ideal ribosomal component to perform such an analysis in germ cells. Instead other proteins such as RPL4/uL4 or RPL9/eL6 are much better suited for this task. Tagged variants of these proteins are well expressed in germ cells, integrated into translating ribosomes and do not influence germ cell functions. Furthermore, germ cell-specific mRNAs are much more efficiently co-purified with RPL4/uL4 and RPL9/uL6 compared to RPL10A/uL1. This study provides a solid basis upon which future germ cell TRAP experiments can be built, and it highlights the need for rigorous testing when adopting such methods to a new biological system.

在许多生物体中，组织复杂性和细胞多样性构成了阻碍我们理解基因表达程序的障碍。为了解决这个问题，已经开发了允许从基因定义的细胞群中轻松分离翻译的 mRNA 的方法。一个突出的例子是翻译核糖体亲和纯化方法，也称为 TRAP。在这里，通过纯化核糖体蛋白 RPL10A/uL1 分离核糖体相关的 mRNA，该蛋白在组织特异性启动子的控制下表达。最初开发用于研究小鼠神经元中的基因表达，现在已被许多不同的生物体和组织采用。有趣的是，TRAP 从未成功地用于分析生殖细胞中的 mRNA 翻译。采用遗传和生化方法相结合，秀丽隐杆线虫种系作为靶组织。令人惊讶的是，我发现 RPL10A/uL1 并不是在生殖细胞中进行此类分析的理想核糖体成分。相反，其他蛋白质如 RPL4/uL4 或 RPL9/eL6 更适合这项任务。这些蛋白质的标记变体在生殖细胞中很好地表达，整合到翻译核糖体中并且不影响生殖细胞功能。此外，与 RPL10A/uL1 相比，生殖细胞特异性 mRNA 与 RPL4/uL4 和 RPL9/uL6 的共纯化效率更高。这项研究为未来的生殖细胞 TRAP 实验奠定了坚实的基础，并强调了在将此类方法应用于新的生物系统时进行严格测试的必要性。