Department of Neurology, University of Michigan, Ann Arbor, MI, USA.
Department of Respiratory Medicine, Xiangya Hospital, Central South University, Changsha 410008, China.
Hum Mol Genet. 2022 Jul 21;31(14):2317-2332. doi: 10.1093/hmg/ddab353.
Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.
重复非 AUG（RAN）翻译 FMR1 5'UTR 中的 CGG 重复产生毒性蛋白，导致脆性 X 相关震颤/共济失调综合征（FXTAS）发病机制。最丰富的 RAN 产物 FMRpolyG 主要在重复上游的 ACG 处起始。FXTAS 患者中缺乏准确的 FMRpolyG 测量。我们使用依赖数据的采集和并行反应监测 (PRM) 质谱联用稳定同位素标记标准肽，在患者样本中鉴定出 FMRpolyG 特征片段。经过免疫沉淀后，PRM 在转染细胞、FXTAS 组织和细胞中检测到 FMRpolyG 特征肽，但在对照中未检测到。我们鉴定出两个氨基末端肽：ACG 起始的 Ac-MEAPLPGGVR 和 GUG 起始的 Ac-TEAPLPGGVR，以及在两个阅读框中 CGG 重复本身内 RAN 翻译起始的证据。细胞应激后所有起始位点增加，eIF1 过表达后减少，并且依赖于 eIF4A 和 M7G 帽。这些数据表明，FMRpolyG 可在人类样本中定量，并且通过类似于近同源密码子的机制以及通过依赖于可用起始因子和细胞环境的过程，在 FMR1 RAN 翻译中起始。
