In vivo analysis of protein translation activity in sea urchin eggs and embryos

Abstract

Protein synthesis is a major regulatory step of gene expression in different physiological processes including development. Translation of proteins in sea urchin is stimulated upon fertilization and is necessary for cell cycle progression and development. Translational control is exerted through multifactorial mechanisms, including mRNA recruitment into polysomes and increased rates of translational activity. In this chapter, we review the methods used in sea urchin eggs and embryos to analyze translation activity in vivo both from perspectives of the proteins and of the mRNAs. First, we describe methods to quantify or visualize newly synthesized proteins with radioactive and non-radioactive labeling techniques. Next we present the polysome isolation and profiling on sucrose gradients, allowing the identification of translated mRNAs. Finally, we outline a procedure to follow the translation of a reporter luciferase protein from an mRNA microinjected into the egg.

Keywords: Luciferase reporter; Microinjection; Polysome gradient; Polysomes; Protein synthesis; Translation; mRNA.