Mapping RNA-Binding Proteins on the Ribosome by Tethered Micrococcal Nuclease

RNA-binding proteins (RBPs) are essential regulators of posttranscriptional gene expression, influencing mRNA processing, translation, and stability. Defining their binding sites on RNA is key to understanding how they assemble into functional ribonucleoprotein (RNP) complexes, but existing footprinting and cross-linking approaches often yield low signal-to-noise, variable efficiency, or require highly purified complexes. To address these limitations, we developed Tethered Micrococcal Nuclease Mapping (TM-map), a sequencing-based strategy that determines the three-dimensional binding sites of RBPs on RNA in vitro. In TM-map, the RBP is fused to micrococcal nuclease (MNase), which upon Ca²⁺ activation cleaves proximal RNA regions, producing fragments whose 3' termini report the spatial proximity of the fusion. We first validated TM-map using the bacteriophage MS2 coat protein bound to its cognate RNA stem-loop engineered into the <i>Escherichia coli</i> ribosome. Cleavage sites mapped proximal to the engineered stem-loop, confirming that tethered MNase reliably reports local protein-RNA proximity on the ribosome surface. We then applied TM-map to the <i>Drosophila</i> Fragile X Mental Retardation Protein (FMRP), a translational regulator with an unresolved ribosome-binding site. Both <i>N</i>- and <i>C</i>-terminal MNase-FMRP fusions produced reproducible cleavage clusters on the 18S rRNA localized to the body and head of the 40S subunit. The similar profiles suggest that FMRP's termini are conformationally flexible and sample multiple orientations relative to the ribosome, consistent with a dynamic interaction rather than a fixed binding mode. TM-map thus provides a simple, proximity-based, and generalizable in vitro approach for visualizing RBP-RNA interactions within native RNP assemblies.