Our assessments of both mRNA and protein copy number combine to suggest that the MBNL family of splicing factors are less abundant in postmitotic endothelial tissue compared with cultured human corneal endothelial cells. Open in a separate window Figure 7 Estimating the quantitative relationship between cellular MBNL protein and the capacity for protein recognition by expanded RNA within intronic TCF4 foci. and human corneal endothelial tissue. Results Using fluorescent in situ hybridization and immunofluorescence, we found that depletion of both MBNL1 and MBNL2 reduces nuclear RNA foci formed by the repeat, suggesting that both are necessary for foci. Quantitative studies of Nivocasan (GS-9450) RNA and protein copy number revealed MBNLs to be abundant in the total cellular pool in endothelial cell lines but are much lower in human corneal endothelial tissue. Studies using human tissue nuclear and cytoplasmic fractions indicate that most Nivocasan (GS-9450) MBNL proteins are localized to the cytoplasm. Conclusions The low levels of MBNL1/2 in corneal tissue, in combination with the small fraction of protein in the nucleus, may make corneal endothelial cells especially susceptible to sequestration of MBNL1/2 by CUG repeat RNA. These observations may explain how a limited number of RNA molecules can cause widespread alteration of splicing and late-onset degenerative FECD. gene (CTG18.1 triplet repeat polymorphism) accounts for up to 70% of FECD cases.7C10 Mutant CUG repeat transcripts accumulate as Rabbit Polyclonal to CPZ nuclear foci in corneal endothelial tissue of affected subjects11,12 without reducing mRNA levels expressed by the parent gene.11,13 These data implicate mutant noncoding regions of RNA as the cause of FECD. The gene encodes the E2-2 protein, a ubiquitously expressed class 1 Nivocasan (GS-9450) basic-helix-loop-helix transcription factor.14 Unlike other trinucleotide repeat diseases, mutant does not cause apparent neurodegenerative disease. However, Nivocasan (GS-9450) neurons and corneal endothelial cells share important similarities that impact our understanding of disease pathology and treatment.15 During embryonic development, corneal endothelial cells are derived from neural crest cells, and adult corneal cells retain peripheral neuronal markers.16 Like neurons, corneal endothelial cells are postmitotic and terminally differentiated. Both neurons and corneal endothelial cells are not replaced, and degeneration slowly degrades function over a patient’s lifetime. There is currently no explanation for the restriction of disease phenotype to corneal tissue in FECD. Myotonic dystrophy type 1 (DM1) is a multisystem disorder caused by a CUG repeat expansion within the 3 UTR of mRNA.17,18 Importantly, this mutation has also been associated with FECD.19,20 This remarkable finding that FECD can be caused by the same expanded repeat within noncoding regions of RNAs associated with two different genes reinforces the conclusion that the mutant expanded CUG repeat RNA is the cause of FECD. A key issue for therapeutic intervention is understanding how mutant RNA molecules can cause a severe degenerative disease. The molecular mechanisms for DM1 have been extensively studied and may offer lessons for understanding FECD. In DM1 cells derived from affected tissues, expanded transcripts accumulate as nuclear foci,21 and the expanded CUG repeat region is thought to sequester muscleblind-like (MBNL) proteins.22C24 MBNL normally acts to regulate splicing, and perturbing the concentration of available MBNL may account for the widespread splicing changes observed in DM1 cells and tissue.25C27 MBNL1 proteins colocalize with the expanded CUG repeat RNA in FECD patient-derived corneal endothelial cells with either or expansions.12,20 Additionally, MBNL2 has been shown to colocalize in cultured endothelial cells of FECD subjects with the expansion.28 In parallel with the suggested mechanism explaining altered splicing in DM1, one hypothesis to explain how RNA might cause FECD suggests that the expanded repeat within the gene binds MBNL proteins and reduces the pool of free cellular MBNL proteins, thereby inducing global splicing changes that ultimately lead to cellular malfunction and degeneration. This hypothesis has been supported by observations that FECD cells or tissue with expansions exhibit changes in the alternative splicing of critical MBNL-sensitive genes relative to normal cells.12,29 Complicating this hypothesis, we previously observed that, in cultured corneal endothelial cells or in tissue, each cell has only a limited number of foci and each focus is a single RNA molecule.30 This observation raised a critical question underlying the mechanism of disease action: how can a small number of mutant RNA molecules affect splicing to cause a late-onset disease? In this report, we characterize MBNL1/2 expression and examine the association of MBNL proteins with mutant RNA in both patient-derived corneal endothelial cell lines and human corneal endothelial tissue. We found that both MBNL1/2 colocalize with the nuclear foci in FECD endothelial tissue. Depletion of MBNL1 or MBNL2 individually did not impact the number of foci in cultured cells. Knockdown of both MBNL proteins resulted in a reduction of foci suggesting that they play a redundant.
Our assessments of both mRNA and protein copy number combine to suggest that the MBNL family of splicing factors are less abundant in postmitotic endothelial tissue compared with cultured human corneal endothelial cells