Localization of mRNAs is a widespread mechanism for dictating where proteins operate in cells and underpins many fundamental processes, from embryonic patterning to synaptic plasticity. This spatial control is mediated by the interaction of 'localization signals' in target mRNAs with RNA-binding proteins (RBPs). These signals frequently lack overt sequence or structural patterns, raising the question of how specificity is achieved. Here we investigate this issue using the Drosophila RBP Egalitarian (Egl), which couples mRNAs to microtubule-based transport through Bicaudal D (BicD) and the dynein motor. We present cryo-electron microscopy structures of Egl-BicD bound to six different RNAs. Egl uses multiple noncanonical double-stranded RNA-binding domains to cooperatively form a recognition pocket around localization signals. Despite substantial variation in length and sequence, each signal adopts a bent stem-loop conformation that, together with base-pair identities at two defined sites, drives Egl engagement. We further demonstrate that Egl dimers couple RNA binding to transport initiation through coincident detection of two RNA elements within the same transcript. Thus, localizing mRNAs are recognized through a combination of shape, positional sequence features and number of structured RNA elements. This work reveals a molecular strategy by which diverse mRNAs can be selectively engaged by a single RBP.
© 2026. The Author(s).