Cytoplasmatic polyadenylation of mRNA by TENT5A is critical for enamel mineralization

Reviewer #1 (Public review):

Summary:

The authors aim to determine whether TENT5A, a post-transcriptional regulator previously implicated in bone formation, also plays a role in enamel development. Using a mouse model lacking TENT5A, they report hypomineralized enamel with structural defects, accompanied by reduced expression, altered poly(A) tail length, and impaired secretion of enamel matrix proteins, particularly amelogenin. By combining ultrastructural imaging, transcriptomics, direct RNA sequencing, and protein localization analyses, the study proposes that TENT5A promotes cytoplasmic polyadenylation and translation of a subset of extracellular matrix transcripts required for enamel biomineralization.

Strengths:

A major strength of this work is its conceptual novelty. To my knowledge, this is the first study to demonstrate that a non-canonical poly(A) polymerase plays a direct role in enamel development, extending post-transcriptional regulation by cytoplasmic polyadenylation from bone to enamel, a biologically distinct and non-regenerative mineralized tissue. The identification of amelogenin as a dominant, tissue-specific target provides a new perspective on how enamel matrix production is regulated beyond transcriptional control.

In addition, the study is supported by a comprehensive and complementary set of approaches linking molecular changes to tissue-level phenotypes. The use of direct RNA sequencing provides strong evidence for selective regulation of poly(A) tail length in specific transcripts rather than global effects on mRNA metabolism, and the phenotypic analyses convincingly connect altered post-transcriptional regulation to defects in enamel structure and mineralization.

Weaknesses:

Although the data support a role for TENT5A in stabilizing and promoting translation of amelogenin and related transcripts, the mechanism underlying substrate specificity remains unresolved. Poly(A) tail length alone does not explain why certain transcripts are regulated while others are not, and the proposed involvement of protein partners or RNA processing steps remains speculative. This limitation should be more clearly framed as an open question rather than an emerging mechanism.

A further limitation is the lack of direct human genetic or clinical evidence linking TENT5A to enamel defects. In humans, loss-of-function variants in TENT5A are known to cause a recessive form of osteogenesis imperfecta, but TENT5A has not been associated with amelogenesis imperfecta or other enamel phenotypes. This limits immediate translational interpretation of the mouse enamel phenotype and highlights the need for future human genetic or clinical studies.

Finally, the manuscript does not address whether other members of the TENT5 family are expressed in ameloblasts or could compensate for the loss of TENT5A, leaving open questions about redundancy and specificity within this family.

Reviewer #2 (Public review):

Summary:

The manuscript by Aranaz-Novaliches describes a study of Tent5a knockout (KO) mice. The authors demonstrate a severe enamel phenotype in these mice, characterized by hypoplastic enamel with markedly disturbed organization of enamel rods. Additionally, they report that Amelx expression is reduced in the mutant compared to wild type (WT) at both mRNA and protein levels. The authors also examine the distribution and co-localization of Amelx and Ambn in ameloblasts and the enamel matrix. These findings are novel and provide important insights into the role of polyadenylation in regulating enamel matrix protein translation and its downstream effects on protein trafficking, secretion, and enamel formation. However, I have multiple concerns regarding the data and its analysis that need to be addressed.

Specific comments:

(1) Introduction

The structure of the introduction is unconventional. The first sentence of the third paragraph states that the goal of this study is to investigate the role of TENT5A in enamel formation, but the rest of the paragraph focuses on enamel in general. The following paragraph claims that the authors discovered the effects of Tent5a deficiency on enamel formation for the first time, yet most of the paragraph discusses enamel proteins and amelogenesis. The choice of references is problematic. The authors cite Sire et al. (2007), which focuses on the origin and evolution of enamel mineralisation genes, a poor fit for this context. A more appropriate source would be a recent review, e.g., Lacruz R et al., Physiol Rev. 2017;97(3):939-993. Ambn constitutes ~5% of the enamel matrix, not 10%. Reference 16 (Martin) is not ideal for murine enamel; more detailed studies exist, e.g., Smith CE et al., J Anat. 2019;234(2):274-290. References on protein-protein interactions (17-19) are also off: Wald et al. studied Ambn-Ambn and Amelx-Amelx interactions separately; Fang et al. focused on Amelx self-assembly only; Kawasaki and Weiss addressed gene evolution. The authors should cite work from Moradian-Oldak's lab, which clearly demonstrates Amelx-Ambn interactions. The last paragraph contains confusing statements, e.g., "TENT5a localized in rER promotes the expression of AmelX and other secreted protein transcripts." Also, the manuscript does not convincingly show disruption of self-assembly beyond overall enamel disorganization.

(2) Results

(a) microCT

Quantitative microCT analyses of WT and KO enamel are needed. At a minimum, enamel thickness and density should be measured from at least three biological replicates per genotype. Severe malocclusion in KO mice is not discussed. The mandibular incisor appears abraded, while the maxillary incisor is overgrown. Is maxillary enamel as affected as mandibular? The age of the mice is not specified. High-resolution scans of isolated mandibular incisors described in Materials and Methods should be included.

(b) SEM

The term "disorganized crystal structure" is incorrect - SEM cannot reveal crystal structure. This requires electron/X-ray diffraction or vibrational spectroscopy. Likely, the authors meant disorganized rods and interrod enamel. The phrase "weak HAP composition" is unclear. Can the increase in interprismatic matrix volume and reduction in rod diameter be quantified? Since rods are secreted by distal Tomes' processes and interrod by proximal Tomes' processes, an imbalance may indicate alterations in the ameloblast secretory apparatus. TEM studies of demineralized incisors are recommended to assess ameloblast ultrastructure.

(c) EMP expression

There is a discrepancy between WB images and data in Figure S2a. In Figure 2b, Amelx band is stronger than Ambn (expected, as Amelx is ~20× more abundant), but in Figure S2a, Ambn appears higher. How was protein intensity in Fig. S2a calculated? Optical density? Was normalization applied? Co-localization in Figure 2d was performed on LS8 cells, which lack a true ameloblast phenotype. Amelx expression in LS8 cells is ~2% of actin (Sarkar et al., 2014), whereas in murine incisors, it is ~600× higher than actin (Bui et al., 2023). Ambn signal is weaker than Amelx, which may affect co-localization results.

(d) Splicing products in Figure 2e

All isoforms except one contain exon 4. The major functional splice product of Amelx lacks exon 4 (Haruyama et al. J Oral Biosci. 2011;53(3):257-266), and there are some indications that the presence of exon 4 can lead to enamel defects. Can it be that the observed phenotype is due to the presence of exon 4?

(e) Co-localization studies

The presented co-localization studies do not demonstrate self-assembly defects; they reflect enamel microstructural defects observed by SEM. Self-assembly occurs at the nanoscale and cannot be assessed by light microscopy except with advanced optical methods. Conclusions based on single images are weak. The authors should perform experiments at least on three biological replicates per genotype, quantify results (e.g., total gray values per ROI of equal pixel size), and use co-localization metrics such as Mander's coefficient. Claims about alternative secretory pathways require much stronger evidence.

The authors should avoid implying that mRNA is inside the ER lumen. It is likely associated with the outer rER surface, which is expected. The resolution of the methods used is insufficient to confirm ER lumen localization.

Reviewer #3 (Public review):

Summary:

It is well established that poly(A) tails at the 3' end of mRNA are critical for mRNA stability, providing another layer of gene regulation. TENT5A is one of the non-canonical poly(A) polymerases that add an extra poly(A) tail. This manuscript demonstrates that the Tent5A mutation leads to mineralization abnormalities in the tooth, shorter poly(A) tails in amelogenin mRNA and some other selected mRNAs, and provides a list of TENT5A interacting proteins.

Strengths:

(1) The authors show in vivo genetic evidence that Tent5a is critical for normal tooth mineralization.

(2) The authors show that the length of the poly(A) tail in amelogenin (AmelX) is 13 bases shorter in Tent5a mutants but not in other mRNAs, such as ameloblastin (Ambn).

(3) Differentially expressed genes (DEGs) in Tent5A mutant tissues (cervical loop) are identified, and some of them show different lengths of poly(A) tails.

(4) TENT5A interacting proteins are identified. Together with the DEGs, these datasets will provide valuable research tools to the community.

Weaknesses:

(1) There is no direct evidence to support the main conclusion; the length of the poly(A) tail is critical for normal tooth mineralization.

(2) The RNAseq data to identify TENT5A substrate is based on the assumption that shorter poly(A) tailed RNA is less stable. However, there are multiple reasons for the differential expression of RNA in Tent5A mutant tissues.

(3) Several TENT5A-interacting proteins have been identified, but, beyond their colocalization with a target mRNA, no mechanistic studies have been conducted.

Author response:

We thank the editors and reviewers for their careful and constructive evaluation of our manuscript. We appreciate the recognition of the conceptual novelty and in vivo relevance of our findings. We have carefully considered all comments and outline below the major revisions and additional analyses we will undertake. For clarity, we address the reviewers’ comments in thematic sections.

Cell-autonomous contribution of Tent5a to phenotype

We agree that the use of a complete knockout model raises the possibility of indirect or non-cell-autonomous effects on tooth development, particularly given the observed dentin alterations. To address this point directly, we are generating and analyzing an ameloblast-specific conditional model we have already on shelf (Ambn-Cre; Tent5a^flox/flox) to determine whether the enamel phenotype arises from cell-autonomous loss of TENT5A in the secretory epithelium. This approach will allow us to distinguish epithelial-intrinsic effects from potential secondary contributions of odontoblasts or mesenchymal tissues. Results from this model will be incorporated into the revised manuscript.

Mechanistic basis and substrate specificity

We agree that the mechanism underlying substrate selectivity of TENT5A requires further clarification. We have performed multiple classical RNA–protein interaction assays, including CLIP-based approaches, without identifying a clear sequence-specific recognition motif. In the revised manuscript, we will present substrate specificity as an open mechanistic question rather than implying a defined recognition mechanism.

To strengthen this aspect, we will extend our analysis to include combined immunoprecipitation strategies and investigation of potential ribosome-associated or co-translational interactions of TENT5A.

In addition, we will further validate selected high-confidence TENT5A interactors identified in our dataset in context of putative changes in AmelX-polyA tail length.

Poly(A) tail length and functional causality

We acknowledge that shortening of the poly(A) tail alone does not formally establish causality. However, our data consistently show that TENT5A-dependent shortening of poly(A) tails correlates with reduced mRNA and protein levels of key enamel matrix components. In the revised manuscript, we will clarify this mechanistic framework more explicitly, integrating poly(A) length, transcript abundance, and protein-level data in a structured manner, while clearly distinguishing correlation from formal proof of causality.

We will also perform additional functional assays, including mRNA stability measurements in vitro in cells with genetic ablation of Tent5a, to further test the link between poly(A) shortening and reduced AmelX protein levels.

Quantitative microCT and enamel morphology

We will include quantitative microCT analyses of enamel thickness and mineral density from multiple biological replicates per genotype (n ≥ 3). Sample numbers will be explicitly stated throughout. Additional high-resolution scans of isolated incisors will be provided. We will also quantify occlusal angle and include whole-skull reconstructions to document malocclusion. Maxillary enamel will be analyzed and quantified alongside mandibular enamel.

SEM terminology will be corrected (e.g., replacing “crystal structure” with “rod/interrod organization”), and structural parameters such as rod diameter and interprismatic matrix proportion will be quantitatively assessed.

We agree that ultrastructural analysis of ameloblast secretory morphology is important. We have experience with TEM analysis of demineralized incisors and will perform additional ultrastructural examination to assess the integrity of Tomes’ processes and the secretory apparatus in Tent5a-deficient ameloblasts. These data will allow us to distinguish between primary alterations in secretory morphology and downstream effects on matrix organization.

Amelx splice variants

We will re-analyze our RNA-seq data with specific attention to exon 4-containing isoforms and clarify the distribution of splice variants in WT and KO samples. These findings will be explicitly discussed in the context of prior literature.

Co-localization and self-assembly claims

We agree that conventional light microscopy cannot directly resolve nanoscale self-assembly events. In Figure 3, our intention was to demonstrate differential subcellular distribution and partial segregation of AMELX and AMBN within secretory compartments, rather than to claim direct visualization of molecular self-assembly. In the revised manuscript, we will clarify this distinction, moderate the terminology accordingly, and provide explicit quantitative co-localization analyses across multiple biological replicates.

TENT5 family paralogs

To address potential redundancy within the TENT5 family, we will analyze published single-cell RNA-seq datasets (Sharir et al., 2019; Krivanek et al., 2020) to assess expression of TENT5 paralogs in ameloblasts. These findings will be validated using targeted transcriptional analyses.

Human clinical relevance

We appreciate the suggestion to examine potential human enamel phenotypes. We will pursue retrospective analysis of clinical and imaging data from patients carrying TENT5A variants through our collaborations with rare disease networks and specialized centers in Europe and the United States. Any relevant findings will be incorporated into the revised manuscript.

Tissue sampling clarification

We apologize for imprecise terminology regarding transcriptomic sampling. The analyzed tissue corresponds to the proximal incisor region up to the mineralization stage. We will include a schematic and clarify nomenclature throughout the manuscript.

Language and data clarity

The manuscript will be thoroughly revised for clarity, consistency of terminology, figure referencing, and accuracy of citations. We will explicitly clarify the methodology used for protein quantification, including normalization strategy and densitometric analysis, to address inconsistencies noted in the supplementary data. We will also expand the discussion to address the biological relevance of moderate poly(A) shortening, referencing established literature demonstrating that even subtle changes in tail length can significantly influence translational efficiency.

Although AMELX is the most abundant enamel matrix protein and exhibits a consistent TENT5A-dependent poly(A) shortening phenotype, our data demonstrate that multiple secreted proteins are similarly affected. We will revise the text to clearly articulate that the enamel phenotype likely reflects the combined contribution of multiple TENT5A-regulated secretory factors rather than a single-gene effect.

We believe these revisions will substantially strengthen the mechanistic, quantitative, and conceptual framework of the study and provide a clearer foundation for interpreting TENT5A-dependent regulation of enamel biomineralization.