Functions of the Tudor-domain containing protein TDRD6 in male germ cells

都铎结构域结合蛋白质中的甲基化精氨酸，并可能结合核酸。在生殖细胞中，几种都铎结构域蛋白的分子和细胞作用尚不清楚，但一些报道指出了其核心和基本功能。TDRD6在精母细胞的粗线期晚期特异性表达。它最初分布在整个细胞中，在减数分裂期间集中在一些分散的颗粒状染色质体中（CB， I型），然后在精子细胞中集中在一个核周CB中（II型）。在第一个资助期，我们确定并表征了TDRD6的两个迄今为止未知的主要功能，它们都与大型蛋白质- rna复合物有关：(i)在无义介导的mRNA衰变（NMD）和（ii）在剪接体组装中的作用。我们最近发表了关于TDRD6在精细胞NMD中的研究（Fanourgakis et al., 2016），提交了关于TDRD6在精细胞剪接中的作用的数据（Akpinar et al., 2016）。最近的数据还表明，TDRD6还存在核定位。根据第一个资助期产生的数据，我们的假设是TDRD6在组装各种rna相关蛋白复合物和含有精氨酸甲基化蛋白的胞内体中起作用。此外，TDRD6通过调节甲基转移酶PRMT5来控制精氨酸甲基化。因此，TDRD6似乎是精子发生过程的中心调节器，该过程依赖于蛋白质精氨酸的对称二甲基化。在申请的第二个资助期，我们计划确定TDRD6在蛋白质复合物组装中的作用及其与PRMT5的关系的基本特性。因此，我们下一个资助期的具体目标是：(1)了解TDRD6的调控(2)了解TDRD6如何控制PRMT5(3)破译TDRD6在细胞核中的作用。我们已经开发了大量的工具和方法来解决这些问题，包括TDRD6缺乏症小鼠，携带全功能TDRD6- lap转基因的小鼠，允许分选特定精母细胞/精母细胞群体的小鼠，以及RIP，染色质体蛋白质组学和转录组学。我们希望获得关于TDRD6和PRMT5的重要见解，这将使我们能够在中期进一步详细研究TDRD6复合物在NMD和剪接中的活性，并确定TDRD6的潜在进一步作用。

Tudor domains bind methylated arginine in proteins and may bind nucleic acids. In germ cells, the molecular and cellular roles of several tudor domain bearing proteins are little understood, yet some reports indicate central and essential functions. TDRD6 is specifically expressed starting in late pachytene in spermatocytes. Initially distributed throughout the cell it concentrates during meiosis in some dispersed granule-like chromatoid bodies (CB; type I), then in spermatids it concentrates in the one perinuclear CB (type II). In the 1st funding period we identified and characterized two hitherto unknown major functions for TDRD6, both relating to large protein-RNA complexes: roles (i) in nonsense-mediated mRNA decay (NMD) and (ii) in spliceosome assembly. Our studies on TDRD6 in spermatid NMD were recently published (Fanourgakis et al., 2016), the data on the role of TDRD6 in spermatocyte splicing are submitted (Akpinar et al., 2016). Very recent data also suggest an additional nuclear localisation of TDRD6. Based on the data generated during the 1st funding period our hypothesis is that TDRD6 acts in assembling diverse RNA-related protein complexes and intracellular bodies that harbor arginine-methylated proteins. In addition, TDRD6 controls arginine methylation itself by regulating the methyl transferase PRMT5. Therefore, TDRD6 appears as a central regulator of processes in spermatogenesis, which depend on symmetric dimethylation of arginine of proteins.In the requested 2nd funding period, we plan to determine fundamental properties of TDRD6 underlying its role in protein complex assembly and its relationship to PRMT5.Our specific aims for the next funding period therefore are:(1) To understand the regulation of TDRD6 (2) To understand how TDRD6 controls PRMT5(3) To decipher the role of TDRD6 in the nucleusWe have developed a plethora of tools and methods to address these questions including TDRD6 deficiency mice, mice carrying a fully functional TDRD6-LAP transgene, mice allowing sorting of specific spermatocyte/spermatid populations, as well as RIP, chromatoid body proteomics and transcriptomics. We expect to obtain important insights into TDRD6 and PRMT5, which will in mid-term allow us to further study in detail the activities of TDRD6 complexes in NMD and splicing as well as to identify potential further roles of TDRD6.