RPL-4 and RPL-9 –Mediated Ribosome Purifications Facilitate the Efficient Analysis of Gene Expression in

摘要

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.