IDENTIFYING MAELSTROM COMPLEXES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 各种类型的转座因子占据了基因组的广阔区域，对基因组的完整性构成了严重的威胁。在包括人类在内的哺乳动物物种中，转座子的负面影响被复杂的细胞机制所抵消，这些机制涉及DNA甲基化和最近发现的称为皮尔纳途径的基于小RNA的机制。这种新的防御机制涉及Argonautes的PIWI亚支的效应蛋白，相关的小RNA和辅助蛋白的干部。帮助皮尔纳产生和功能的此类蛋白质之一是Maelstrom（MAEL），其是一种进化上保守的蛋白质，具有非经典HMG盒核酸结合结构域和未知生化功能的推定RNAseH折叠结构域。我们先前的遗传和细胞生物学研究已经证明了小鼠MAEL对于皮尔纳途径的稳健活性的重要作用。为了进一步阐明皮尔纳在小鼠雄性生殖细胞谱系中的生物发生和功能机制，我们提出系统地表征MAEL相关蛋白质组和RNA组。 为此，我们将进行亲和纯化的MAEL含复合物，确定MAEL相互作用蛋白（MIPs）的质谱，并通过酵母双杂交，免疫荧光分析，并在体外结合的方法与MAEL的相互作用的特点。我们还将确定小的和大的RNA组成的MAEL含复合物，其亚细胞分布特征的RNA荧光原位杂交在野生型和Mael突变生殖细胞，并确定其表达的影响的Mael突变。这些方法的组合将为皮尔纳在小鼠雄性生殖系中的生物发生和功能提供重要的新线索。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Various classes of transposable elements occupy vast genomic territories and pose significant ganger to the genomic integrity. In mammalian species, including humans, the negative effect of transposons is countered by sophisticated cellular machinery that involves DNA methylation and a recently discovered small RNA-based mechanism known as the piRNA pathway. This novel defensive mechanism involves effector proteins of the PIWI subclade of Argonautes, associated small RNAs and a cadre of auxiliary proteins. One of such proteins aiding in piRNA production and function is Maelstrom (MAEL), an evolutionarily conserved protein with a non-canonical HMG box nucleic acid binding domain and a putative RNAseH fold domain of unknown biochemical function. Our prior genetic and cell biological studies have demonstrated the essential role of mouse MAEL for the robust activity of the piRNA pathway. To further elucidate the mechanism of piRNA biogenesis and function in the mouse male germ cell lineage, we propose to systematically characterize MAEL-associated proteome and RNAome. To this end, we will perform affinity purification of MAEL-containing complexes, identify MAEL-interacting proteins (MIPs) by mass spectrometry, and characterize their interactions with MAEL by means of yeast two hybrid, immunofluorescent analysis, and in vitro binding approaches. We will also identify small and large RNA composition of MAEL-containing complexes, characterize their subcellular distribution by RNA fluorescent in situ hybridization in wild type and Mael mutant germ cells, and determine the effect of the Mael mutation on their expression. The combination of these approaches will provide essential new clues about piRNA biogenesis and functioning in the mouse male germline.