RNA-binding proteins in eye development and disease: implication of conserved RNA granule components

The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1, and Bmp4 are commonly required for their development. In contrast, our understanding of posttranscriptional regulation in eye development and disease, particularly regarding the function of RNA-binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila as well as several vertebrate models such as fish, frog, chicken, and mouse. Interestingly, of the 42 RBPs that have been investigated for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as processing bodies, stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate posttranscriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2, and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly, and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving posttranscriptional regulatory networks in eye development and disease. WIREs RNA 2016, 7:527-557. doi: 10.1002/wrna.1355 For further resources related to this article, please visit the WIREs website

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. 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

Mechanical properties of <i>G</i>. <i>vaginalis</i> colonized cervices.

<p>Area (A), Max Load (B), Stiffness (C), Max Stress (D), Modulus (E), and Max Strain (F) are shown for both control and <i>G</i>. <i>vaginalis</i> colonized cervices. All data is presented as means with standard deviations and significance noted at p< 0.05, (n = 10–11 in each group).</p

<i>G</i>. <i>vaginalis</i> increases soluble E-cadherin in the CV space.

<p>Levels of soluble E-cadherin were measured by ELISA. T-test analysis with Welch’s correction was calculated for significance of protein expression between the groups (***p<0.0001). Values are mean ± SD.</p

<i>G</i>. <i>vaginalis</i> increases levels of IL-6 in the cervicovaginal space and amniotic fluid.

<p>Levels of IL-6 in the CVF (<b>A)</b> and in the AF <b>(B)</b> were measured via ELISA. T-test analysis with Welch’s correction was performed to determine statistical significance between two groups (***p = 0.0007 in the CVF and ***p = 0.0008 in the AF analysis) (N = 8 Control and N = 12 <i>G</i>. <i>vaginalis</i> group). Values are mean ± SD.</p

Mechanical properties of <i>G</i>. <i>vaginalis</i> colonized cervices.

<p>Area (A), Max Load (B), Stiffness (C), Max Stress (D), Modulus (E), and Max Strain (F) are shown for both control and <i>G</i>. <i>vaginalis</i> colonized cervices. All data is presented as means with standard deviations and significance noted at p< 0.05, (n = 10–11 in each group).</p

<i>G</i>. <i>vaginalis</i> colonization of the CV space of timed-pregnant CD-1 mice.

<p>Quantification of the 16S gene of <i>G</i>. <i>vaginalis</i> in the CVF of animals inoculated with 5X10⁸ CFU/mL, was performed via qPCR using a specific <i>G</i>. <i>vaginalis</i> 16S probe. Graphs shows the average quantity mean detected by qPCR of N = 8 Control and N = 12 <i>G</i>. <i>vaginalis</i> group. T-test analyses with Welch’s correction between these groups was performed (***p<0.0001). Values are mean ± SD.</p

Increased expression of Tff-1 in the cervix of <i>G</i>. <i>vaginalis</i> colonized animals.

<p>Increased expression of Tff-1 in the cervix of <i>G</i>. <i>vaginalis</i> colonized animals.</p

Increased gene expression of IL-1β, IL-8 and IL-10 in the cervix of <i>G</i>. <i>vaginalis</i> colonized animals.

<p>Gene expression levels of IL-8 (A), IL-1-β (B), IL-10 (C), and TNF-α (D) were measured by QPCR. T-test with Welch’s correction was performed in each group (N = 8 Control and N = 11 <i>G</i>. <i>vaginalis</i> group) (**IL-8 (p = 0.0055),*IL-1-β (p = 0.0120) and *IL-10 (p = 0.0140)). Values are mean ± SD.</p