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Inez Oh, Xiaodong Zhang, Nicholas Tran, Shiming Chen; Hyper-stability of mutant mRNA causes pathogenic overexpression of mutant CRX in autosomal dominant retinopathies. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3590.
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Mutations in the cone-rod homeobox gene CRX have been associated with autosomal dominant (ad) retinopathies, but the underlying disease mechanisms are poorly understood. Class III frameshift human CRX mutations create premature termination codons (PTCs), producing truncated proteins that interfere with wild-type (WT) CRX function in heterozygotes. Animal models of Class III CRX mutations (E168d2, E168d2neo and Tvrm65 mice, and Rdy cats) develop retinopathies, and heterozygotes display elevated ratios of mutant to WT mRNA and protein, correlating with phenotype severity. We hypothesize that PTCs expose a stability code within the Crx mRNA 3’ untranslated region (3’UTR), producing hyper-stable mutant RNA, which amplifies mutant protein toxicity, leading to global mis-regulation of gene expression and photoreceptor degeneration. To test this hypothesis, the half-life of WT and mutant Crx mRNA was measured and 3’UTR regions that regulate Crx mRNA stability identified.
In retinal explants from P14 WT (C57BL/6J) or homozygous Crx-E168d2 mice, the half-life of Crx mRNA was assessed by measuring Crx mRNA levels (qRT-PCR) at various times after 1) inhibiting RNA synthesis with Actinomycin D, or 2) metabolic pulse-chase [5’-bromouridine-immunoprecipitation-chase (BRIC)]. Critical RNA regions that confer hyper-stability were identified by in vitro reporter gene assays in HEK293 cells transfected with serial Crx-3’UTR deletions.
Both RNA decay assays revealed at least 2-fold increases in the half-life of mutant mRNA relative to WT, confirming the hyper-stability of mutant mRNA. This was attributed to the coding region-derived UTR sequence (cUTR) resulting from the PTC. Increased length of the cUTR (due to early PTC) correlates with enhanced mRNA stability, recapitulating in vivo observations from the E168d2 (early PTC) and Tvrm65 (late PTC) mouse models.
The pathogenic overexpression of mutant CRX stems from a novel mRNA stabilization mechanism intrinsic to specific RNA regulatory sequences. Our findings establish a new paradigm for investigating mis-regulation of gene expression in neurological diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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