Cryo-EM structures of Egl–BicD–RNA complexes reveal how diverse mRNAs are selected for subcellular localization

Localization of mRNAs is a widespread mechanism for controlling where proteins operate in cells and underpins many fundamental processes, from embryonic patterning to synaptic plasticity. This spatial control is mediated by RNA-binding proteins (RBPs) that interact with ‘localization signals’ within target mRNAs. However, these signals lack overt sequence or structural patterns, even when targeted by the same protein, raising the question of how specificity is achieved. Here, we investigate this question using the Drosophila RBP Egalitarian (Egl), which couples developmentally important mRNAs to microtubule-based transport via Bicaudal D (BicD) and the dynein motor protein. We present cryo-EM structures of Egl– BicD in complex with six different RNAs. Egl uses multiple non-canonical double-stranded RNA-binding domains to cooperatively form a recognition pocket around a localization signal. 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 binding. We further demonstrate that Egl dimers couple RNA binding to transport initiation through coincident detection of two RNA elements within the same transcript. Collectively, we show that diverse localizing mRNAs are recognized through a combination of shape, positional sequence features, and number of structured RNA elements. This work provides a framework for understanding how other RBPs engage highly variable mRNAs.