Ott Kristen M, Nguyen Tram, Navarro Caryn
Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts 02118 Graduate Program in Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118.
Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts 02118.
G3 (Bethesda). 2014 Sep 19;4(11):2247-57. doi: 10.1534/g3.114.014332.
Transposable selfish genetic elements have the potential to cause debilitating mutations as they replicate and reinsert within the genome. Therefore, it is critical to keep the cellular levels of these elements low. This is especially true in the germline where these mutations could affect the viability of the next generation. A class of small noncoding RNAs, the Piwi-associated RNAs, is responsible for silencing transposable elements in the germline of most organisms. Several proteins have been identified as playing essential roles in piRNA generation and transposon silencing. However, for the most part their function in piRNA generation is currently unknown. One of these proteins is the Drosophila melanogaster DExH box/Tudor domain protein Spindle-E, whose activity is necessary for the generation of most germline piRNAs. In this study we molecularly and phenotypically characterized 14 previously identified spindle-E alleles. Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain. Additionally, we found that processes that depend on piRNA function, including Aubergine localization, Dynein motor movement, and retrotransposon silencing, were severely disrupted in alleles with DExH box domain mutations. The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much. From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.
可转座自私遗传元件在基因组内复制和重新插入时，有可能引发使人衰弱的突变。因此，将这些元件在细胞内的水平维持在较低水平至关重要。在生殖细胞系中尤其如此，因为这些突变可能会影响下一代的生存能力。一类小的非编码RNA，即Piwi相关RNA，负责在大多数生物体的生殖细胞系中沉默可转座元件。已经鉴定出几种蛋白质在piRNA的产生和转座子沉默中发挥着重要作用。然而，在很大程度上，它们在piRNA产生中的功能目前尚不清楚。其中一种蛋白质是果蝇的DExH盒/Tudor结构域蛋白纺锤体-E（Spindle-E），其活性对于大多数生殖细胞系piRNA的产生是必需的。在本研究中，我们对先前鉴定的14个纺锤体-E等位基因进行了分子和表型特征分析。在表达可检测到的纺锤体-E蛋白的等位基因中，我们发现有5个在DExH盒结构域发生了突变。此外，我们发现依赖于piRNA功能的过程，包括茄子蛋白（Aubergine）定位、动力蛋白运动和逆转座子沉默，在具有DExH盒结构域突变的等位基因中受到严重破坏。许多这些等位基因的表型与最强的纺锤体-E表型一样严重，而在纺锤体-E其他区域发生突变的等位基因对这些过程的影响则没有那么大。从这些数据我们得出结论，纺锤体-E的DExH盒结构域对于其在piRNA途径和逆转座子沉默中的功能是必需的。
