Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI, USA.
Department of Neurology, University of Michigan, Ann Arbor, MI, USA.
EMBO Rep. 2019 Sep;20(9):e47498. doi: 10.15252/embr.201847498. Epub 2019 Jul 25.
A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.
CGG 三核苷酸重复扩张位于 FMR1 的 5'UTR 中，导致神经退行性疾病脆性 X 相关震颤/共济失调综合征（FXTAS）。该重复支持一种称为重复相关、非 AUG（RAN）翻译的非典型蛋白质合成方式。CGG 重复中 RAN 翻译的机制尚不清楚。为了鉴定 RAN 翻译的调节剂和潜在的治疗靶点，我们在基于细胞的测定和 FXTAS 的黑腹果蝇模型中对真核起始因子和 RNA 解旋酶进行了基于候选的筛选。我们从 FMR1 5'UTR 中的扩展 CGG 重复中鉴定出了多个毒性和 RAN 翻译的调节剂。这些包括 DEAD 盒 RNA 解旋酶 belle/DDX3X、解旋酶辅助因子 EIF4B/4H 以及起始密码子选择性因子 EIF1 和 EIF5。在体内和培养的人细胞中，破坏 belle/DDX3X 选择性地抑制了果蝇中的 FMR1 RAN 翻译，并减轻了果蝇和原代啮齿动物神经元中的重复诱导毒性。这些发现表明 RNA 二级结构和起始密码子保真度是介导 FMR1 RAN 翻译的关键因素，并确定了治疗重复相关神经退行性疾病的潜在靶点。
