A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome

RNA exosomopathies, a growing family of tissue-specific diseases, are linked to missense mutations in genes encoding the structural subunits of the conserved 10-subunit exoribonuclease complex, the RNA exosome. Such mutations in the cap subunit gene EXOSC2 cause the novel syndrome SHRF (Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies). In contrast, exosomopathy mutations in the cap subunit gene EXOSC3 cause pontocerebellar hypoplasia type 1b (PCH1b). Though having strikingly different disease pathologies, EXOSC2 and EXOSC3 exosomopathy mutations result in amino acid substitutions in similar, conserved domains of the cap subunits, suggesting that these exosomopathy mutations have distinct consequences for RNA exosome function. We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by introducing the EXOSC2 mutations in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells have defects in cell growth and RNA exosome function. We detect significant transcriptomic changes in both coding and non-coding RNAs in the rrp4 variant, rrp4-G226D, which models EXOSC2 p.Gly198Asp. Comparing this rrp4-G226D mutant to the previously studied S. cerevisiae model of EXOSC3 PCH1b mutation, rrp40-W195R, reveals that these mutants have disparate effects on certain RNA targets, providing the first evidence for different mechanistic consequences of these exosomopathy mutations. Congruently, we detect specific negative genetic interactions between RNA exosome cofactor mutants and rrp4-G226D but not rrp40-W195R. These data provide insight into how SHRF mutations could alter the function of the RNA exosome and allow the first direct comparison of exosomopathy mutations that cause distinct pathologies.RNA外切酶体病（RNA exosomopathies）是一类不断扩大的组织特异性疾病家族，与保守的10亚基核糖核酸外切酶复合物（即RNA外切酶体（RNA exosome））的结构亚基编码基因所发生的错义突变密切相关。帽状亚基基因EXOSC2上的此类突变可引发新型综合征SHRF（全称："Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies"，即身材矮小、听力损失、色素性视网膜炎与特殊面容综合征）。与之相反，帽状亚基基因EXOSC3的外切酶体病突变会导致1b型桥小脑发育不全（PCH1b）。尽管二者引发的疾病病理特征差异显著，但EXOSC2与EXOSC3的外切酶体病突变均会导致帽状亚基相似的保守结构域内发生氨基酸替换，这提示此类外切酶体病突变对RNA外切酶体功能的影响存在差异。我们以酿酒酵母（Saccharomyces cerevisiae，以下简称S. cerevisiae）为模型，通过在其同源基因RRP4中引入EXOSC2的突变，构建了首个SHRF致病性氨基酸替换的体内模型。所得的rrp4突变体细胞存在细胞生长与RNA外切酶体功能缺陷。我们在模拟EXOSC2 p.Gly198Asp突变的rrp4-G226D突变体中，检测到编码RNA与非编码RNA均出现显著的转录组变化。将该rrp4-G226D突变体与此前已报道的EXOSC3 PCH1b突变酿酒酵母模型rrp40-W195R进行对比，发现这两种突变体对特定RNA靶标的效应存在显著差异，这为引发不同病理的外切酶体病突变存在不同机制后果提供了首个证据。一致的是，我们检测到RNA外切酶体辅助因子突变体与rrp4-G226D之间存在特异性的负向遗传相互作用，而与rrp40-W195R则无此现象。这些数据为阐明SHRF突变如何改变RNA外切酶体功能提供了见解，同时首次实现了引发不同病理的外切酶体病突变的直接对比。