Department of Biochemistry and Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, United States of America.
PLoS Biol. 2011 Apr;9(4):e1001052. doi: 10.1371/journal.pbio.1001052. Epub 2011 Apr 26.
FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.
FUS/TLS 是一种核酸结合蛋白，当其发生突变时，可能导致一部分家族性肌萎缩侧索硬化症（fALS）。虽然 FUS/TLS 通常主要位于细胞核内，但致病性突变形式的 FUS/TLS 会转移并在受影响的脊髓运动神经元或神经胶质细胞的细胞质中形成包含物。在这里，我们报告了一种人类 FUS/TLS 表达的酵母模型，该模型再现了致病突变蛋白病理学的多个显著特征，包括核质易位、包含物形成和细胞毒性。蛋白结构域分析表明，FUS/TLS 的羧基端（大多数与 ALS 相关的突变聚集于此）是必需的，但不足以引起蛋白毒性。利用酵母过表达文库进行的全基因组遗传筛选鉴定了五个酵母 DNA/RNA 结合蛋白，它们由酵母基因 ECM32、NAM8、SBP1、SKO1 和 VHR1 编码，这些蛋白可以挽救人类 FUS/TLS 的毒性，而不改变其表达水平、细胞质易位或包含物形成。此外，hUPF1，ECM32 的人类同源物，也可以挽救该模型中 FUS/TLS 的毒性，验证了酵母模型，并暗示 RNA 加工或 RNA 质量控制机制可能存在不足，是 FUS/TLS 介导的毒性的机制之一。检查 FUS/TLS 表达对酵母中选定 mRNA 衰减的影响表明，无意义介导的衰减途径可能不是毒性或抑制的主要决定因素。
