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Abstract

Translation – the process by which mRNAs are decoded into nascent peptides – is fundamental to life. This process is regulated by various RNA-binding proteins (RBPs), which interact with their target mRNAs. While traditional biochemical approaches have provided valuable insights into translational control, they rely on ensemble measurements of bulk mRNAs in test tubes. Consequently, the behavior of individual mRNAs and their spatiotemporal dynamics in cells during translational control remain largely unexplored. Offering a way to address such limitations, we developed a method for imaging translational control by Argonaute (AGO) proteins, a class of RBPs, at single-mRNA resolution in cells. This method, which employs three-color fluorescence microscopy to detect mRNAs, nascent peptides, and AGO, can also serve as a versatile platform for analyzing translational control by other RBPs. In this protocol, we provide a step-by-step guide for implementing this method to facilitate spatiotemporal studies of translational control at the single-cell and single-mRNA levels.