Stemness factor Mex3a times translation and protein trafficking to ensure robust differentiation of olfactory sensory neurons

  1. Martín Escamilla del Arenal
  2. Lauren C. Tang
  3. Albana Kodra
  4. Hani Shayya
  5. Aileen Ugurbil
  6. Olga Stathi
  7. Keskin Abdurrahman
  8. Adan Horta
  9. Joan Pulupa
  10. Junqiang Ye
  11. Marko Jovanovic
  12. Stavros Lomvardas
  13. Rachel Duffié

  • Curated by eLife

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    eLife Assessment

    This important study identified Mex3a protein with dual RNA-binding protein/ubiquitin ligase function as a pivotal regulator of olfactory sensory neurons (OSN) differentiation and lineage fidelity. The authors employed a combination of systems biology approaches (e.g., single-cell RNA sequencing, proteomics) and newly developed animal models (e.g., HyperTRIBE) to provide solid evidence that abrogation of Mex3a disrupts cilia structure and polarity of OSNs. Notwithstanding that this article is of a broad potential interest across different biomedical disciplines ranging from RNA to developmental biology, additional mechanistic data connecting identified Mex3a mRNA targets and ensuing OSN phenotypes would further strengthen this study.

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Abstract

During the switch from progenitor to differentiated cell, cellular physiology must change to accommodate increased translation and trafficking of membrane-bound proteins. We identify RNA-binding and E3 ubiquitin ligase Mex3a as a key driver of proper neuronal differentiation by regulating mRNA translation and trafficking of cell surface proteins in the context of Unfolded Protein Response (UPR) signaling. Loss of Mex3a in immature olfactory sensory neurons (OSNs) leads to defects in cilia structure, cell surface protein expression, and planar cell polarity in mature OSNs. Proteomics reveal a Mex3a-dependent decrease in proteins related to vesicle transport, lipid metabolism, and ribosome biogenesis. We identify RNA and ubiquitin targets of Mex3a and provide evidence that Mex3a may confer K27 ubiquitin linkage on substrates. Finally, modulating cellular levels of Mex3a changes the recruitment of translation factors Serbp1 and p-eEF2 to ribosomes with possible effects on translation. Our data reveal how a stemness factor regulates development post-transcriptionally and post-translationally to ensure robust differentiation.

Highlights

Loss of stemness factor Mex3a in immature olfactory neurons leads to defects in mature olfactory neurons.

Translation/Trafficking of cell surface proteins, cilia structure, and planar cell polarity are compromised in the absence of Mex3a.

Mex3a may confer K27 ubiquitination on stress granule protein Serbp1 and ribosome protein Rps7.

Mex3a levels are associated with Serbp1 and p-eEF2 recruitment to ribosomes.

Article activity feed

  1. eLife

    eLife Assessment

    This important study identified Mex3a protein with dual RNA-binding protein/ubiquitin ligase function as a pivotal regulator of olfactory sensory neurons (OSN) differentiation and lineage fidelity. The authors employed a combination of systems biology approaches (e.g., single-cell RNA sequencing, proteomics) and newly developed animal models (e.g., HyperTRIBE) to provide solid evidence that abrogation of Mex3a disrupts cilia structure and polarity of OSNs. Notwithstanding that this article is of a broad potential interest across different biomedical disciplines ranging from RNA to developmental biology, additional mechanistic data connecting identified Mex3a mRNA targets and ensuing OSN phenotypes would further strengthen this study.

  2. eLife

    Reviewer #1 (Public review):

    The study by Escamilla del Arenal et al. utilized a conditional knockout mouse model to study the role of Mex3a in immature olfactory sensory neurons (OSN). Mex3a is a dual-functional protein that has RNA-binding function and ubiquitin-E3 ligase activity. The results revealed that Mex3a expression is critical for proper OSN differentiation and contributes to cell surface protein trafficking and translation, cilia structure, and planar cell polarity in mature neurons. Moreover, Mex3a enforces lineage fidelity, selectively repressing sustentacular programs in neurons and neuronal programs in sustentacular cells.

    In addition, the authors established an in vivo HyperTRIBE mouse model to identify Mex3a RNA targets and incorporated UbiFast into the Mex3a conditional knockout (cKO) model to find its protein targets to investigate how Mex3a regulates OSN differentiation. The experimental systems are laborious and comprehensive, which allowed the authors to identify new Mex3a putative targets in OSN.

    The phenotypic results derived from the conditional Mex3a cKO mice are solid. Mechanistic findings also revealed that, in addition to facilitating protein degradation, Mex3a may confer K27 ubiquitin linkage on its target proteins, which has a non-proteolytic role but affects target protein activity, other post-translational modifications, or protein-protein interactions. However, among all Mex3a putative targets, the authors decided to emphasize on the Mex3a-mediated K27 ubiquitination on stress granule protein Serbp1 and ribosome protein Rps7, and the association between Mex3a expression and Serbp1 and p-eEF2 ribosome recruitment. This Mex3a-Serbp1-p-eEF2 ribosome recruitment axis, although it can be important in Unfolded Protein Response (UPR) signaling, seems rather general and cannot explain the striking lineage-specific phenotypes observed in the mouse model. The authors need to provide more solid evidence to demonstrate that K27-Ubiquitinylation of Serbp1 is a key step of Mex3a function in OSN differentiation to strengthen the relation between the phenotypes and mechanism presented in this study.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    In this manuscript, Arenal and colleagues demonstrate that loss of Mex3a leads to defects in cell surface protein trafficking, translation, ciliary structure, and planar cell polarity in mature neurons. Through proteomic analyses, the authors show that Mex3a depletion alters the abundance of proteins involved in vesicular transport, lipid metabolism, and ribosome biogenesis. Using the HyperTRIBE approach, the authors further identify targets of Mex3a and provide evidence supporting a role for K27-linked ubiquitination in regulating these substrates. Mechanistically, the study suggests that Mex3a levels influence the recruitment of SERBP1 and phosphorylated eEF2 (p-eEF2) to ribosomes, contributing to translational repression.

    Strengths:

    Overall, this is a very interesting and well-written manuscript that significantly advances our understanding of Mex3a function and its role in neuronal development, particularly in olfactory sensory neurons. The data are clearly presented and thoughtfully interpreted.

    Weaknesses:

    I have a few minor comments that may further strengthen the manuscript and improve its accessibility to a broader readership.

    (1) In Figure 3B, the authors describe Mex3a localization to cytoplasmic granules. However, it is unclear how these compartments were defined. It would strengthen the conclusions if the authors included co-localization experiments using established cytoplasmic granule markers (e.g., stress granule markers) to define the identity of these structures more precisely. This would clarify whether Mex3a associates with stress granules, RNA processing bodies, or another class of ribonucleoprotein granules.

    (2) Functional validation of K27-linked ubiquitination on SERBP1
    To further define the functional significance of K27-linked ubiquitination, it would be informative to mutate the relevant lysine residue(s) on SERBP1 and examine whether this alters its recruitment to ribosomes or affects translational repression. Such an experiment would provide more direct evidence that K27-linked ubiquitination of SERBP1 mediates the observed translational effects.

    (3) Discussion of vesicular trafficking and lipid metabolism targets
    The identification of Mex3a targets involved in vesicular trafficking and lipid metabolism, including COPII coat components such as Sec31a and lipid regulatory proteins such as Sec14 and PIP5K1A, is particularly intriguing. The authors may wish to expand the Discussion to address how regulation of these proteins could contribute to defects in plasma membrane trafficking and planar cell polarity. Integrating these findings with the observed cell surface trafficking phenotypes would further enhance the mechanistic framework of the study.

  4. eLife

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, the authors investigate the role of the KH and RING domain-containing protein Mex3a in the differentiation and maturation of olfactory sensory neurons. Using conditional knockout of Mex3a in immature neurons, they show that mature olfactory sensory neurons display defects in membrane protein trafficking, including olfactory receptors and Adcy3, together with abnormalities in ciliary radial organization and planar cell polarity. Through single-cell RNA sequencing and quantitative proteomics, the authors further show that Mex3a-deficient neurons fail to properly resolve the unfolded protein response and exhibit transcriptomic features suggestive of lineage mixing with sustentacular cells. The study also introduces a methodological advance by adapting HyperTRIBE for use in transgenic mice, which enables the identification of in vivo Mex3a RNA targets, including components of Wnt signaling that appear to be under translational repression by Mex3a. The authors then pursue one of these targets to further explore the role of Mex3a in translational repression.

    Strengths:

    First, it addresses an important biological and conceptual question. Mex3a is a multifunctional protein with the potential to couple RNA regulation, protein homeostasis, and key cellular processes, yet its in vivo role in neuronal differentiation remains poorly understood. By focusing on Mex3a in olfactory sensory neurons, the manuscript asks a timely and important question of how post-transcriptional regulation contributes to the maturation of highly specialized neurons, including the establishment of ciliary architecture, membrane protein trafficking, and cell polarity. Second, the generation and validation of an inducible in vivo mouse HyperTRIBE system represents a technical advance. By incorporating the Adar deaminase domain into a transgenic mouse model, the authors establish a rigorous and useful approach for identifying Mex3a RNA targets in vivo, which is likely to be valuable to the wider RNA biology community. Third, the study integrates the Mex3a knockout model with single-cell RNA sequencing, quantitative mass spectrometry-based proteomics, ubiquitin profiling, and ribosome-related analyses, providing a broad and multilayered view of the Mex3a knockout phenotype. Finally, the imaging analyses revealing altered ciliary content and organization in olfactory sensory neurons identify an interesting and potentially important link between Mex3a, cilia biology, and vesicular trafficking. More broadly, the manuscript reflects a very substantial experimental effort, and each individual dataset has the potential to be useful for the field.

    Weaknesses:

    A main weakness of the manuscript is that the mechanistic links between the major findings remain somewhat correlative, and the biological narrative is not fully sustained through the later figures. The study documents defects in membrane trafficking, ciliary radial organization, and planar cell polarity, and it identifies candidate targets with clear relevance to these processes, including factors linked to vesicle trafficking. However, the manuscript then shifts its mechanistic focus toward translational regulators such as Serbp1 and Rps7, without adequately connecting these later analyses back to the core phenotypes established earlier. As a result, there is a noticeable disconnect between the phenotypic emphasis of the study and the mechanistic validation that follows.

    A second weakness is that, given the breadth and potential importance of the datasets generated, validation remains limited for several of the major conclusions. This reduces confidence in the interpretation of the single-cell, proteomic, ubiquitin-related, and ribosome-associated analyses, and also limits the future value of these datasets as a resource for the field. Because the manuscript aims to address several major questions at once, stronger validation and clearer integration across the different experimental arms are needed for the conclusions to feel fully supported.

    Finally, the HEK293T overexpression experiments are less solid than the in vivo analyses and do not provide equally strong support for the proposed mechanisms. In this context, some of the observed effects on cytoskeletal organization, membrane-less granule formation, and ribosome profiles may be indirect, which makes it difficult to weigh these findings alongside the much stronger in vivo phenotypes.

  5. Version published to 10.64898/2025.12.26.696633 on bioRxiv