A platform for analyzing translational control by RBPs at single-mRNA resolution in cells

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.

Caption of Graphical Abstract

The graphical abstract shows the workflow of this method. Reporter mRNAs are used to image single mRNAs, nascent peptides, and the RBP of interest in cells (top). These images are then processed by quantitative 3D image analysis, including outline detection, spot detection, and colocalization analysis, which allows in situ quantification of the translational status and RBP-binding status of all mRNA molecules (bottom). This approach enables the analysis of translational control by RBPs at single-mRNA resolution in cells.