Barnum CE et al. (2020), The Tudor-domain protein TDRD7, mutated in cong...

XB-ART-56985

Hum Mol Genet 2020 Jul 29;2912:2076-2097. doi: 10.1093/hmg/ddaa096.

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The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology.

Barnum CE , Al Saai S , Patel SD , Cheng C , Anand D , Xu X , Dash S , Siddam AD , Glazewski L , Paglione E , Polson SW , Chuma S , Mason RW , Wei S , Batish M , Fowler VM , Lachke SA .

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Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7-/- mice. Early postnatal Tdrd7-/- animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7-/- lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7-/- cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7-/- lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7-/- cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7-/- lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7-/- fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7-ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7's novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

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Species referenced: Xenopus laevis
Genes referenced: cdkn2b cdkn2c cdknx cryba1 hspb1 npat slc7a5 tcf3 tdrd7
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???displayArticle.disOnts??? cataract 36 [+]
???displayArticle.omims??? CATARACT 36; CTRCT36
Phenotypes: Xtr Wt + hspb1 MO (Fig 11 B, C row2,3, col 2,4, D E F)

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	Figure 1. Phenotypic characterization of Tdrd7−/− lens defects. (A) Light microscopy-based examination of ocular defects in postnatal (P) day 90 Tdrd7−/− mouse lenses. The control eye exhibits a normal iris and a clear lens (left panels), while Tdrd7−/− eye shows a flattened iris (top right panel, indicated with asterisk) and a smaller lens with posterior rupture and cataract (bottom right panel indicated with asterisk). Note: the dissected lenses in the bottom panel are balanced on their equator. (B) Histological analysis of control and Tdrd7−/− eye tissue at embryonic (E) 16.5 and postnatal (P) 4 and P30 stages. While the control lenses show no defects through P30, Tdrd7−/− lenses exhibit profound lens defects with 100% penetrance at P30. (C) Tdrd7−/− mice display visible eye and lens defects by P22. Light microscopy of control and Tdrd7−/− eye and lens indicates no discernable difference at P18. However, at P22, Tdrd7−/− mice exhibit a flattened iris (asterisk) and cataract (asterisk, right image middle panel) at 100% penetrance. Furthermore, lens defects in Tdrd7−/− mice are also detected by histological analyses at P22.
	Figure 2. RNA-Seq analysis of Tdrd7−/− lens. (A) Bioinformatics pipeline for analyzing RNA-Sequencing data; delineating various steps for quality control, adaptor removal, sequence integrity, sequence mapping, estimating counts per transcript and differential analysis. (B) Gene ontology (GO) analysis of 49 differentially expressed genes with fold change (FC) >1.5 and FPKM >10 expression in lens tissue. (C) GO analysis of 85 differentially expressed genes with FC >1.5 and FPKM > 5 expression in lens tissue. (C) GO analysis of 168 differentially expressed genes with FC >1.5 and FPKM > 2 expression in lens tissue.
	Figure 3. iSyTE-based analysis of Tdrd7−/− lens RNA-Seq data identifies Hspb1 among the high-priority candidates. (A) Flowchart of analysis of RNA-Seq data for the identification of high-priority candidate genes in Tdrd7−/− lens. (B) An integrated analysis using iSyTE lens expression and enrichment data along with functional annotation and GO enrichment of significant (FC ±1.5, P-value ≤ 0.05) RNA-Seq differentially expressed genes (DEGs). GO categories for DEGs are indicated on the left. iSyTE-based expression and enrichment data at normal embryonic and postnatal lens development stages (E10.5, E12.5, E16.5 and P0) for the Tdrd7−/− lens DEGs are represented in the heat map diagram using ‘red–white–green’ and ‘red–white–white’ color gradients, respectively. Lens expression of DEGs at early postnatal stage P0 over that at early embryonic stage E10.5 (expression in late versus early lens) is represented using ‘blue–white–yellow’ color gradient. (C) Pearson correlation heat map of candidate genes based on iSyTE wild-type lens expression data across the four developmental stages. The integrated approach outlined in (A–C) applies GO category enrichment as well as iSyTE-based analysis of DEGs and associates Hspb1 as a top priority candidate gene (criteria: expression score in normal lens >2000, enrichment score in normal lens > 4.0, differential expression in Tdrd7−/− lens >1.5-fold change, P-value <0.05). (D) RT-qPCR analysis of Hspb1 validates its downregulation in Tdrd7−/− lenses at stages P4 and P15. Asterisk indicates P-value <0.01.
	Figure 4. 2D-DIGE proteome screen identifies HSPB1 protein to be downregulated in Tdrd7−/− lens. (A and B) 2D-DIGE analysis showed that protein coinciding with ‘spot 951’ was downregulated in Tdrd7−/− lens. (C) Abundance of spot 951 (later identified as HSPB1 protein) was significantly reduced in P15 Tdrd7−/− lens (asterisk represents P-value of 0.039). (D) Spot 951 and a reference spot (later identified as CRYBA1) was picked up for LC-MS/MS-based identification. (E) LC-MS/MS identified spot 951 as mouse HSPB1 with a 100% protein CI as well as a 100% Best Ion CI, while the reference spot was identified as CRYBA1. (F) Western blot confirmed the reduction of HSPB1 protein in Tdrd7−/− lenses compared to control.
	Figure 5. Scanning electron microscopy demonstrates Tdrd7−/− lenses have abnormal fiber cell morphology. Scanning electron microscopy (SEM) was performed to visualize cortical fiber cells for control and Tdrd7−/− lenses at stages P18 and P28. While the control appeared normal, abnormal fiber cell morphology (asterisk) was observed in Tdrd7−/− lenses at both P18 and P28.
	Figure 6. Phalloidin staining for F-actin demonstrates abnormal cellular morphology specifically in fiber cells after nuclear degradation in Tdrd7−/− lenses. (A) Diagram of a lens (left) with the cross-sectional plane (red) through the lens equator. Schematic of the resulting lens equatorial cross section (right) wherein epithelial cells are located at the periphery and hexagonal morphology of fiber cells is visualized. Early to late maturation stages of differentiating fiber cells are indicated, along with the organelle-free zone. (B) Control and Tdrd7−/− lens sections at P15 were stained with phalloidin to visualize F-actin. DNA was visualized by Hoechst stain. Images from comparable areas representative of early to late fiber differentiation and maturation (left to right) are shown for control and Tdrd7−/− lenses. There is no discernable difference in young differentiating and maturing lens fiber cells (left-most panel and left-half of the second panel) between control and Tdrd7−/− lenses. However, coinciding with nuclear degradation (asterisk), fiber cells in Tdrd7−/− lens showed abnormal F-actin distribution, indicative of abnormal fiber cell morphology as compared to control (second panel). (C) Zoom-in of middle image panels shows abnormal fiber cell morphology and F-actin distribution coinciding with nuclear degradation (asterisk) and beyond in Tdrd7−/− lens.
	Figure 7. WGA and F-actin staining demonstrates abnormal membrane morphology and fiber cell organization specifically in fiber cells with nuclear degradation in Tdrd7−/− lenses. Control and Tdrd7−/− lens sections at mouse stage P15 were stained with wheat germ agglutinin (WGA) and phalloidin to visualize cellular membrane and F-actin, respectively. Hoechst stain was used to visualize DNA. Images from comparable areas representative of early to late fiber differentiation and maturation (left to right) are shown for both control and Tdrd7−/− lenses. While there is no discernable difference in young differentiation and maturing lens fiber cells at the lens periphery (left-most panel and left-half of the second panel) between control and Tdrd7−/− lenses, fiber cells showed abnormal WGA staining and abnormal fiber cell morphology in the Tdrd7−/− lens, coinciding with nuclear degradation (arrowheads), compared to control (second panel).
	Figure 8. HSPB1 protein staining in fiber cells increases during normal lens development but is reduced in Tdrd7−/− lens fiber cells. (A) To examine HSPB1 protein expression in normal mouse lens development, immunostaining with HSPB1 antibody was performed on sagittal sections of wild-type mouse embryonic lenses E12.5 and E14.5 and postnatal lenses at P0 and P15. HSPB1 is progressively expressed in lens fiber cells during development (asterisk). (B) Cross sections of control and Tdrd7−/− P15 lenses were immunostained with HSPB1 antibody. Reduction in HSPB1 protein staining in Tdrd7−/− fiber cells that have lost their nuclei is indicated by asterisk. DNA (blue) is stained by DAPI. Scale bar indicates 20 μm. Abbreviation: e, anterior epithelium of the lens; f, fiber cells; r, retina.
	Figure 9. TDRD7 protein associates with Hspb1 mRNA in the lens. RNA immunoprecipitation (RIP) analysis using TDRD7 antibody was performed for wild-type mouse lenses at stage P15. This was followed by (A) RT-PCR and (B) RT-qPCR analysis, both of which demonstrate that Hspb1 mRNA is enriched in TDRD7 pulldowns compared to control. Asterisk indicates P-value <0.05.
	Figure 10. Single-molecule fluorescence in situ hybridization (smFISH) coupled with immunostaining demonstrates TDRD7 protein to co-localize with Hspb1 mRNA in lens fiber cells. (A) Wild-type mouse lens at stage P15 was stained with complementary RNA probes specific to Hspb1 mRNA (green), (B) along with immunostaining for TDRD7 protein (red). (C) Merged image of the co-staining (yellow) and (D) analysis of significant co-localization of Hspb1 mRNA and TDRD7 protein using custom-written MATLAB program as described in Methods (colored open circles). (A’-D”) shows zoom-in of regions indicated by broken-line boxes in A–D. Arrows indicate co-localizing elements scored by the MATLAB analysis.
	Figure 11. Hspb1 knockdown in X. tropicalis causes eye and lens defects. (A) Flowchart of X. tropicalis assay and phenotypic scoring. (B) Representative images of un-injected and injected side of X. tropicalis embryos injected with either control MO, Hspb1 MO or Hspb1 MO together with mouse Hspb1 mRNA (mHspb1). (C) Zoom-in images of white-dotted area in (B). (D) Chart showing the distribution of ocular defects in X. tropicalis embryos injected with either control MO, Hspb1 MO or Hspb1 MO together with mHSPB1 mRNA. The number of embryos for the different conditions is indicated on top. Black asterisk represents significant differences in the observed number of eye defects between the different conditions. For the control MO versus Hspb1 MO comparison, the P-value is 4.54 × 10−6, and for the Hspb1 MO versus Hspb1 MO + mHspb1 mRNA comparison, the P-value is 1.38 × 10−6. The control MO versus Hspb1 MO + mHSpb1 mRNA comparison is not significant (P-value = 0.55). (E) Histology analysis-based images and (F) DAPI-stained images of sections of un-injected and Hspb1 MO injected X. tropicalis embryos. White asterisk represents small eye/lens defects in Hspb1 MO-injected side of embryos. Black asterisks in (C), (E) and (F) represent rescue of eye defects in Hspb1 MO + mHspb1 mRNA-injected side of embryos. In (E) and (F), the lens is indicated by a white-dotted area.
	Figure 12. Models for TDRD7 function in lens development and cataractogenesis. The following models are proposed to explain the temporal and the cell and molecular basis of the lens defects and cataract observed in Tdrd7−/− mice. (A) Overlay of temporal aspects of Tdrd7−/− lens defects, at a molecular and phenotypic level, corresponding to wild-type expression of the TDRD7 target mRNA Hspb1, a heat shock protein associated with F-actin under stress conditions, in normal mouse lens development. iSyTE microarray-based expression analysis of wild-type lenses shows that mRNA expression of Hspb1 upregulates before slight downregulation, but yet maintaining relatively high levels in postnatal wild-type lens development. Interestingly, Tdrd7−/− lenses show reduced levels of Hspb1 mRNA at early postnatal stages (RNA-Seq, RT-qPCR data), several days prior to morphological detection of the lens cellular defects. Furthermore, HSPB1 protein levels remain low in Tdrd7−/− lenses in later postnatal stages at P15 (proteome, western, immunofluorescence data), just prior to the initial detection of the defect at the cellular level (SEM at P18) or the gross morphological level (overt cataract at P22). The timing of expression of the lens defects in Tdrd7−/− mice coincides with the timing of normal upregulation of Hspb1 in wild-type postnatal lens development and therefore may reflect the disruption of the expression dynamics of this F-actin-associated protein upon TDRD deficiency. (B) Based on the current molecular and phenotypic data, including the critical observation that Tdrd7−/− mice show abnormal cellular morphology particularly in fiber cells after nuclear degradation, the following model is proposed. The association of TDRD7 protein with Hspb1 mRNA ensures its elevated levels, which translate into high levels of HSPB1, a heat shock molecular chaperone protein necessary for F-actin stability in cells subjected to stress. TDRD7-based elevated levels of HSPB1 protein may function in the maintenance of normal F-actin distribution and cellular morphology in late maturation stage fiber cells, which can be considered a stress-like condition as they undergo nuclear degradation.

References [+] :

Agrawal, Compound mouse mutants of bZIP transcription factors Mafg and Mafk reveal a regulatory network of non-crystallin genes associated with cataract. 2015, Pubmed