A Single-Molecule RNA Mobility Assay to Identify Proteins that Link RNAs to Molecular Motors

mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules, we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosin or kinesin through their well-characterized interacting proteins results in quantitative differences in RNA mobility. This methodology provides a framework for identifying RBPs that mediate associations with motors.