TDRD7 mutations in human pediatric cataract. (A) Cataract in DGAP186 (left eye, white arrowhead). (B) Ideogram of normal and inverted chromosome 9 [inv(9)]. Inversion breakpoints are shown by red lines, with a schematic below of TDRD7. Dotted black line marks breakpoint that disrupts TDRD7 within the 2.6-kb region shown and in TDRD7 protein. The black bar indicates TDRD7 genomic probe in Southern analysis. TaqI and StuI refer to the fragments resulting from restriction enzyme digest. The red bar indicates fosmid clone G248P8912G10 used as a TDRD7-specific probe. Exons are indicated by purple boxes. (C) Chromosomal spread of DGAP186 lymphoblastoid cells analyzed by fluorescence in situ hybridization shows split TDRD7-specific red probe, whereas green anchor probe remains intact in inv(9). White arrowheads indicate inverted genomic region that hybridizes to TDRD7-specific probe; white arrow represents noninverted region (see fig. S1). (D) TDRD7 haploinsufficiency is demonstrated by Western blot of DGAP186 lymphoblastoid cells. (E) Pedigree of consanguineous family F3R with congenital cataract (solid symbols, affected status; half-solid symbols, carrier status). (F) Sequence chromatogram shows c.1852_1854del (p.617delVal) mutation (boxed) in family F3R (see fig. S2).

Tdrd7 deficiency causes cataract and glaucoma. (A) In situ hybridization of E12.5 mouse embryo (coronal section) demonstrates Tdrd7 RNA expression in differentiating lens fiber cells (FC) (arrowhead), and its absence in the AEL (arrow). (B) In situ of stage 21 chick embryonic eye (coronal section) shows TDRD7 expression in lens FCs (arrowhead) and absence in AEL. NR, neuroretina; RPE, retinal pigment epithelium. (C) Cataract (white arrowhead) in chick lenses infected with TDRD7-shRNA RCAS virus. (D) Absence of cataract in 1-month-old Tdrd7 heterozygous mouse (control) and (E) cataract in age-matched Tdrd7 null mouse lens. (F) Histology of control lens at 3 months shows no ocular abnormality and (G) a severe cataract in Tdrd7 null mouse lens at same age. Note vacuoles in FC lens compartment (arrow) and rupture of lens capsule that extrudes fiber cell mass (arrowhead) into the vitreous. RD, mild to severe retinal dysplasia, focally present in some mutants. (H) Tdrd7 null (red diamonds), but not control (blue triangles), mice exhibit age-dependent increase in IOP (see fig. S5). (I and J) Histology of iridocorneal angles showing normal morphology and open configuration (*) in both control (I) and mutant (J) eyes. Mutant angles are largely normal and open, but some mutants had mild, focal abnormalities of Schlemm’s canal (SC) and trabecular meshwork (TM). Iris strands that attach to the cornea are common in mice (arrow) but are not continuous around the angle and do not block drainage. Angles of all histologically assessed mice with high IOP at 6 to 12 months were open (n = 3). Co, cornea; Ir, iris. (K to N) At ages >20 months, the optic nerve head and nerve fiber layer (RGC axons, arrows) in control mice (K) appear normal, whereas Tdrd7 null mice (L) have considerable nerve damage with essentially no nerve fiber layer (arrows) and optic nerve head excavation (*). V, vessel. (M and N) Retinal periphery of control mouse (M) has normal cell density (arrows) in the ganglion cell layer (GCL), whereas mutant (N) with glaucomatous excavation of the nerve head has obvious cell loss. INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: [(F) and (G)] 500 μm; [(I) and (J)] 50 μm; [(K) and (L)] 100 μm; [(M) and (N)] 50 μm.

TDRD7 RNA granules interact with STAU1-RNPs in lens fiber cells. (A) Immunofluorescence (IF) of mouse E12.5 lens (coronal section) with TDRD7 antibody demonstrates highly specific punctate expression in lens FCs and absence in AEL. (B) Higher magnification of E12.5 lens FCs stained with TDRD7 antibody shows numerous cytoplasmic granules (arrowheads). (C) Costaining with SYTO RNASelect reveals a similar pattern of cytoplasmic RNA granules (arrowheads). (D) 4′,6′-diamidino-2-phenylindole (DAPI) staining in same section. (E) Merged image [(B) to (D)] reveals colocalization (yellow) of TDRD7 with cytoplasmic RNA granules (see fig. S7). RNA staining of TDRD7 granules is somewhat variable and may reflect dynamic interactions of these granules. Scale bars: (A) 15 μm; (E) 2.5 μm. (F) IF of mouse E12.5 lens shows numerous cytoplasmic granules stained with antibodies against DCP1A (red) and TDRD7 (green). Colocalized granules appear yellow. (Inset) Higher magnification demonstrates colocalization of DCP1A P-bodies and TDRD7 RNA granules. (G) Staining of E12.5 lens with STAU1 antibody reveals numerous granules (red) in FC cytoplasm. These granules colocalize (yellow) to a high degree with TDRD7 granules (green). (Inset) Higher magnification indicates intimate colocalization (yellow, white arrowheads) of TDRD7 and STAU1 granules. (H) At E11.5, TDRD7 and STAU1 expression show a high degree of co-localization (yellow) in the anterior portion of the FC compartment. DAPI-stained nuclei are blue. (Inset) Higher magnification demonstrates high degree of colocalization (yellow) of TDRD7-RGs and STAU1-RNPs. Scale bars: (F) 15 μm; (F inset) 4 μm; (G) 15 μm; (G inset) 1 μm; (H) 10 μm; (H inset) 4 μm.

Tdrd7 is critical for RNA granule formation and HSPB1 expression. (A) SRA01/04 cell line shows increased numbers of P-bodies (detected by DDX6/RCK antibody) and stress granules (detected by elF3b antibody) in response to oxidative stress (sodium arsenite, 0.1 mM). The numbers of stress granules are markedly reduced, and P-bodies are modestly reduced in TDRD7 knockdown SRA01/04 cells (for quantification, see fig. S14). Scale bar: 5 μm. (B) (Upper panels) IF with TDRD7 antibody demonstrates robust TDRD7-RGs in Tdrd7 heterozygous mouse lens and absence in Tdrd7 null mouse mutant lens. Scale bar: 3 μm. (Lower panels) IF with HSPB1 antibody demonstrates reduction of HSPB1 protein in P4 Tdrd7 null lens (see fig. S15). Scale bar: 20 μm. EZ, equatorial zone; OFZ, organelle-free zone.

TDRD7 regulates mRNAs critical to lens development and RG function. (A) Microarray analyses of Tdrd7-KD 21EM15 mouse lens cells reveals down-regulation of RNA granule genes and human cataract genes and up-regulation of other genes normally down-regulated during fiber cell differentiation. Genes are assigned to specific classes, denoted I to V. Jag1 is expressed in fiber cells but is highly restricted. (B) Microarray analyses of mouse Tdrd7 null mutant lens at P4 and P30 reveals down-regulation of RNA granule genes and human cataract genes. Fold changes (FC) are expressed as Log₂(FC). Error bars in (A) and (B) represent propagated SEM. (C) RIP using TDRD7 antibody, followed by RT-PCR, reveals Crybb3, Hspb1 enriched in TDRD7 complexes. (D) Model for TDRD7 function in lens development. In lens fiber cells, TDRD7-RGs regulate certain key lens transcripts (e.g., Crybb3 and Hspb1) directly via binding, and others indirectly via interactions with STAU1-NPs, which also bind specific transcripts (e.g., Sparc and Epha2) directly (26). These interactions may allow TDRD7, alone or in conjunction with STAU1, to protect specific mRNAs from the RNA decay mechanisms that normally exist in lens fiber cells, thus selectively stabilizing these mRNAs for translating the high levels of refractive proteins needed for lens transparency.