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Inez Oh, Shiming Chen; Global RNA metabolic changes associated with a dominant-negative Crx mutation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1003.
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Mutations in the cone-rod homeobox gene CRX are associated with autosomal dominant retinopathies but the underlying disease mechanisms are poorly understood. Frameshift human CRX mutations create premature stop codons, producing truncated proteins that interfere with wild-type (WT) CRX function in heterozygotes. We previously reported a mouse model of such a mutation, Crx-E168d2. The heterozygous E168d2/+ mouse shows early severe photoreceptor deficits resembling human disease, correlating with elevated ratios of mutant:WT mRNA and protein. We hypothesized that hyper-stability of E168d2 mRNA underlies the toxic accumulation of its mRNA and protein. Further, RNA-binding proteins essential for RNA stability show altered expression in diseased retinas, raising the possibility that E168d2 misregulates genes by affecting both mRNA synthesis and degradation. This study aimed to develop a high-throughput technology to measure global RNA synthesis and decay rates in normal and diseased retinas to determine if E168d2 disrupts these processes.
Thiol(SH)–linked alkylation for the metabolic sequencing of RNA (SLAMseq) was performed on retinal explants from P14 WT or E168d2/+ mice to measure RNA synthesis rates by monitoring 4-thiouridine (4sU) incorporation into newly synthesized RNA over 2 hours (h), and RNA decay rates by an 8h pulse-chase after 2h 4sU-labeling. Chemical conversion of 4sU allows detection of labeled RNA by deep-sequencing. Subsequent computational analysis profiles differences in synthesis and decay rates of individual transcripts within and between samples.
SLAMseq successfully detected 4sU incorporation into newly synthesized RNA species. Among these, photoreceptor (PR)-specific transcripts (e.g. Rho, Crx, Nrl) were enriched relative to housekeeping (HK) genes (e.g. Actb, Gapdh) in WT retinas, suggesting that PR genes are more actively transcribed than HK genes. RNA decay rate shows similar differences between PR and HK genes. The fast turnover of PR-specific transcripts is consistent with the biology of metabolically active PRs. RNA synthesis and decay rates were altered for a number of genes in E168d2/+ vs. WT retinas, supporting our hypothesis that E168d2 affects both processes.
SLAMseq is useful for studying RNA metabolism under normal and disease conditions. Understanding the effect of altered RNA metabolism on gene misregulation could reveal gene therapy targets.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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