GSE134384
The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology
Mutations of the RNA-granule component TDRD7 (OMIM: 611258) cause pediatric cataract in humans. Here, we applied an integrated approach to elucidate the molecular pathology of cataract in Tdrd7 targeted-knockout (Tdrd7-/-) mice. Tdrd7-/- animals precipitously develop lens fiber cell abnormalities early in life, suggesting a global-level breakdown/mis-regulation of key cellular processes. High-throughput RNA-sequencing followed by iSyTE-integrated bioinformatics-based analysis identified the molecular chaperone and cytoskeletal-modulator, HSPB1 (HSP27), among the high-priority down-regulated candidates in Tdrd7-/- lens. Moreover, a protein 2-D fluorescence difference gel electrophoresis-coupled mass spectrometry screen also identified HSPB1 to be reduced in Tdrd7-/- lens, offering independent support for focusing efforts on this factor to explain Tdrd7-/- cataract. Reduction of HSPB1 preceded lens morphological abnormalities, suggesting that cytoskeletal defects underlie the Tdrd7-/- cataract phenotype. In agreement, scanning electron microscopy revealed abnormal fiber cell membrane protrusions in Tdrd7-/- lenses. Significantly, abnormal F-actin staining was detected specifically in Tdrd7-/- fiber cells that exhibit nuclear degradation, thereby revealing that there are distinct mechanisms based on pre- or post-nuclear degradation differentiation stage for F-actin cytoskeletal maintenance in fiber cells. Further, 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 a specific pre-nuclear degradation area of differentiating fiber cells, indicating that TDRD7-ribonucleoprotein complexes are necessary for controlling optimal levels of key factors in lens development. Together, these data uncover a novel role for TDRD7 in regulating elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts. Overall design: For RNA-sequencing (RNA-Seq) experiments, mouse lenses at stage P4 (n = 15 lenses per biological replicate) were collected from  Tdrd7-/- or control mice. RNA isolation was performed using the mirVanaTM RNA isolation kit (Life Technologies, Grand Island, NY). Total RNA isolation, followed by removal of small molecular weight RNA was performed according to manufacturer's instructions. Library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina), and sequencing was performed on a 2x75 paired end run using standard protocols on an Illumina HiSeq 2500 sequencing system. Briefly, mRNA was purified from the total RNA samples using Oligo dT conjugated magnetic beads, converted to adaptor-tagged, paired-end fragments which were then used for cluster generation onto a TruSeq v3 flow cell according to the Illumina® TruSeq™ RNA Sample Preparation Kit v2. Sequencing was carried out using the SBS Sequencing Kit. Images were analyzed using the Illumina Pipeline software (version RTA 1.13.48/CASAVA 1.8.2), and bases were called and translated to generate FASTQ sequence files.