Tent5a KO mice displays amelogenesis imperfecta and Tent5a expression in teeth.

(A) 3D-micro-computed tomography (µCT) images of representative WT (up row) and Tent5a KO mice (lower row). Mouse head showing broken and hypomineralized incisor and phase contrast μCT images of transversal and Longitudinal sections of mouse incisor teeth. Green-to-yellow pseudocolor bars represent the increasing level of mineralization. Scale bar, respectively, 1mm, 250μm and 500μm. (B) SEM images of a mouse incisor cross-section displaying WT and Tent5a KO enamel. Enamel thickness and crystallite organization is altered in Tent5a KO samples. Scale bar, respectively, 100μm and 2.5μm. (C) In situ hybridization of Tent5a mRNA showing expression in secretory ameloblasts in mouse incisor teeth. Scale bar, 200µm.

AmelX mRNA is polyadenilated by TENT5A.

(A) mRNA levels of AmelX and Ambn in mice ameloblasts normalized to Rpl19 measured by RT-qPCR. Data bars are mean±SEM. Statistical tests are unpaired student t-tests, *p-value <0.05, **p-value <0.01. (B) Western blot analysis of AMELX and AMBN protein levels in WT (lane 1) and Tent5a KO (lane 2) teeth. (C) Direct RNA sequencing (DRS)-based poly(A) length profiling of AmelX mRNA isolated from incisor cervical loop, p-value < 0.001. (D) TENT5A protein and AmelX and Ambn mRNA localization in LS8 ameloblast-like cells. Scale bar, 10 μm. Colocalization parameters were measured using Manders’ coefficient, statistical test Non-parametric Paired Wilcoxon, ****p-value < 0.0001. (E) All AmelX isoforms scheme and comparison of poly(A) tail length in WT and Tent5a KO ameloblast measured by Nanopore DRS.

Immunostaining of AMELX (red) and AMBN (green) in WT and Tent5a KO mouse ameloblasts.

(A) Enamel organic matrix in the extracellular region of maturation stage ameloblasts and high magnification image of enamel. Scale bar, respectively, 20μm and 5μm. (B) Intracellular region of secretory stage ameloblasts cells. Scale bar, respectively, 20μm and 10μm. (C) Histogram of colocalization of AMELX and AMBN signal intensities in secretory vesicles. Quantification was measured from the straight line marked in intracellular ameloblasts. (D) Maturation stage intracellular region of ameloblasts. Scale bar, respectively, 20μm and 10μm.

Identification of TENT5A substrates in ameloblasts by transcriptomics assays.

(A) Illumina sequencing mRNA transcriptomics heatmap comparing WT and Tent5a KO cervical loop samples. Relevant dysregulated genes for Amelogenesis are presented. N=3, p-value <0.05. (B) RT-qPCR analysis of enamel proteins in mice ameloblasts normalized to Rpl19. Data presented as mean ± SD. Statistical tests are unpaired student t-tests, *p-value p<0.05, **p-value p<0.01. (C) DRS of WT and Tent5a KO cervical loop RNA. MA-like plot showing transcripts with a significant difference in poly(A) tail length. Adjusted p-value < 0.05, length difference >5, at least 10 reads in WT sample. (D) Distribution of poly(A) tail length of mRNAs encoding extracellular matrix and other proteins in WT and Tent5a KO ameloblasts. (E) A PCR-based PAT assay shows the poly(A) tail length of AmelX, Ambn and Clu transcripts in ameloblasts. Lanes 1, 4 and 7 are gene-specific PCR products, lanes 2, 5 and 8, WT ameloblast poly(A) tail PCR product and lanes 3, 6 and 9 Tent5a KO samples.

TENT5A subcellular localization and potential interacting partners identification.

(A) Immunofluorescence staining of ameloblast immortalized cell line IMAL and LS8 cell line displays Calnexin ER protein (green) and Tent5a_Myc (red). Scale bar, 10μm. (B) Volcano plot mass spectrometry analysis of potential TENT5A interacting protein partners in ameloblast cells. P-value <0.03. (C) Subcellular localization of potential TENT5A interacting partners using Human cell map and subcellular compartment analysis from mass spectrometry data. P-value <0.03. (D) FISH assay showing AmelX and Ambn mRNA localization with Calnexin ER protein in WT and Tent5a KO ameloblasts primary cells. Scale bar, 10μm. Colocalization parameters were measured using Manders’ coefficient, statistical test Non-parametric Paired Wilcoxon test,p-value*<0.001.