The structure of Egalitarian in complex with the K10 mRNA localization signal reveals a modular binding surface required for function

Reviewer #1 (Public review):

Summary:

The authors sought to define the molecular mechanism by which the adaptor protein Egalitarian (Egl) recognizes and binds specific mRNA localization signals -- in particular, the K10 transport and localization signal (TLS) -- to initiate dynein-based transport in Drosophila. In doing so, they identified the minimal Egl domains required for RNA binding, determined the atomistic structure of the Egl-RNA complex, and explored the recognition mechanism (shape vs. structure). They furthermore performed in vivo functional validation using CRISPR-mediated genome editing in Drosophila that showed that the identified binding residues are biologically essential.

Strengths:

The authors provided a detailed crystal structure of the Egl-RNA complex at high resolution. In particular, they used a MBP-fusion crystallization driver to be able to resolve the flexible C-terminal domain of Egl (EHD). The authors' use of an integrative approach combining X-ray crystallography with binding assays and in vivo functional validation provides compelling evidence for their claims.

The work provides a detailed interaction mapping that identifies the protein residues responsible for the electrostatic interaction with the RNA. In doing so, the work explains how Egl can recognize diverse RNA sequences by demonstrating that Egl binds primarily to the phosphate backbone and specific structural bulges, providing a plausible model for how one protein can recognize many different localization signals that share little sequence similarity.

Weaknesses:

Discrepancy in the stoichiometric Egl-to-RNA ratio (the structural data in the paper indicate a 1:1 ratio, whereas previous single-molecule transport studies suggest a 2:1 ratio) remains unanswered, with the likely explanation that the truncated version of the protein might not capture the full (native) assembly. While the authors acknowledge this in the Discussion, the paper would benefit from this issue being raised earlier, already in the Results section. Moreover, there is a notable omission of a recent preprint on a very similar topic [https://www.biorxiv.org/content/10.1101/2025.08.02.668268v1.full].

In vitro, Egl shows a relatively high affinity for non-target RNAs such as the MS2 loop, whereas it is highly selective in vivo. Is it possible that other cofactors are required for the high-fidelity sorting not present in the study? Testing binding in the presence of co-factors (BicD or Dlc) could indicate whether they increase the specificity for target RNAs over non-target ones.

Including a more diverse set of size-matched RNA controls would have significantly strengthened the paper's claims regarding specificity. Using RNAs that mimic K10 TLS would have provided a more rigorous test of the shape-recognition by Egl - using, for instance, decoy RNAs of the same length but with differently positioned bulges (or no bulges at all) or testing other known localization signals (like bicoid or hairy) of similar length.

Appraisal of aims:

The authors successfully determined the crystal structure of the Egl-RNA complex, identifying a modular binding surface composed of the EXO domain, a helical linker, and the EHD. They effectively demonstrated that Egl uses a combination of shape-specific recognition (targeting RNA bulges) and sequence-specific interactions (bonding with specific bases), and confirmed the biological necessity of these findings by showing that mutating the identified residues in living flies leads to infertility and oocyte differentiation defects. These results provide robust evidence for the authors' claims that they have defined a minimal RNA localization signal. In particular, the correlation between the L-Triple mutation's binding defect and its total sterility in flies provides proof that the identified binding surface is the functional one. While the 1:1 stoichiometry remains a point for further investigation, the authors transparently address that full-length transport may require a 2:1 assembly, suggesting their structure represents the fundamental building block of that larger complex.

Impact of the work on the field:

This study provides a high-resolution picture of how a dynein adaptor recognizes its cargo. It moves the field from predictive models to atomic-level certainty, setting a benchmark for studying other similar transport complexes. By proving that Egl recognizes RNA shape (bulges) as much as sequence, the work changes the outlook on the search for localization signals in other genomes, moving beyond simple sequence motifs to 3D structural signatures. The coordinates deposited in the EBI (IDs: 9UJU, 9UJY, 9UUG) provide a resource for the modelling of higher-order transport complexes. The identification of specific residues (e.g., the L-Triple) provides the community with tools to disrupt RNA transport in Drosophila without destroying the entire protein, allowing for more nuanced studies of development.

Reviewer #2 (Public review):

Summary:

Hong et. al. aimed to elucidate the structural basis of the Egalitarian recognition of the K10 mRNA. Using X-ray crystallography and several biochemical, biophysical, and cellular techniques, they were able to shed light on the formation, stability, and basis of interaction of the complex. The authors successfully accomplished their goal.

Strengths:

The experiments are well-performed and convincing. The manuscript is well-written.

Weaknesses:

(1) Some statistical analysis would improve the manuscript. In particular, the manuscript has several results that are based on comparisons, such as Kd. Adding p-values for significance is recommended, and this would improve the treatment of data.

(2) When showing interactions (dotted lines) in structural figures, adding the distance would be useful and is recommended.

(3) Additional SI Figure. It would enrich the manuscript to have the composite simulated annealing-omit 2|Fo| - |Fc | electron density maps for the structures contoured at a given sigma, superimposed on the final refined model. This would represent how well the data fits into the model.

Author response:

We would like to thank the editors and the reviewers for their thoughtful and constructive assessment of our manuscript. We appreciate the reviewers' positive recognition of our research and their thoughtful assessment of our data.

In the upcoming revision, we will incorporate rigorous statistical analysis (p-values) for our binding assays, optimize the structural figures and summary tables for better clarity, and discuss the recent preprint paper alongside the nuances of Egl-BicD stoichiometry. Regarding the suggestion for CLIP-seq, we agree that a global analysis would be a valuable extension of this work. However, as our lab’s core expertise is in structural biology, and the in vivo functional studies in this manuscript were conducted through a collaboration to validate our structural findings, we feel that such a large-scale genomic study falls beyond the scope of the current structural report.