RNA Editing in the Neonatal and Adult Testis

描述(申请人提供)：RNA编辑，RNA物种内核苷酸的修饰，在哺乳动物中广泛存在。RNA编辑具有改变蛋白质编码转录本的编码能力以及修改靶向转录本的转录后调控的能力。高通量RNA测序的最新进展显著增加了RNA编辑的已知目标的数量，以及在其中观察到RNA编辑的组织。不幸的是，对于许多系统来说，RNA编辑的目标和结果并不清楚。这项工作的目标是确定RNA编辑在男性生育中的作用，特别是减数分裂后生殖细胞的发育。先前的工作已经证明，由于减数分裂后生殖细胞的缺陷，推测的RNA编辑调控因子的全局消融会导致男性不育。这项工作的长期目标是从功能上表征RNA编辑在男性生育中的作用，并确定异常的RNA编辑是否会导致某些男性不育。每七对夫妇中就有一对受到不孕不育的影响，大约有一半的夫妇 因男性因素缺陷造成的病例。在这些人中，又有近一半是特发性的，不孕不育的分子基础尚不清楚。使用RNA编辑酶的全局和特定于生殖细胞的敲除小鼠模型，这项提议将检验生殖细胞RNA编辑导致生殖细胞蛋白质组变化和对男性生育能力至关重要的假设。目的1是确定先前描述的假定的RNA编辑调节子的全局丢失是否改变了睾丸中RNA编辑的频率和/或位置分布。目标2将确定在生殖细胞发育和生育中是否需要关键的RNA编辑酶，并确定该酶的生殖细胞特异性丢失是否会改变睾丸RNA编辑。最后，目标3将通过质谱学检测编辑的转录本编码的多肽以及编辑的转录本相对于其未编辑的转录本的多聚体结合，确定在mRNA编码区的RNA编辑是否影响蛋白质编码能力或编辑目标的翻译。从这些研究中获得的知识对男性生殖和RNA编辑领域做出了实质性的贡献，并可能为特发性男性不育的机制提供洞察。

DESCRIPTION (provided by applicant): RNA editing, the modification of nucleotides within an RNA species, is widespread in mammals. RNA editing has the ability to alter the coding capacity of protein coding transcripts as well as modify the post- transcriptional regulation of targeted transcripts. Recent advancements in high throughput RNA sequencing has substantially increased the number of known targets for RNA editing as well as the tissues in which RNA editing has been observed. Unfortunately, the targets and result of RNA editing is unclear for many systems. The goal of this work is to define the role of RNA editing in male fertility, specifically the development of post- meiotic germ cells. Previous work has demonstrated that global ablation of a putative RNA editing regulator results in male infertility due to defects in post-meiotic germ cells. The long-term goal of this work is to functionally characterize the role of RNA editing in male fertility and determine if abnormal RNA editing is causative to some male infertility. Infertility affects one in seven couples, roughly half of these cases being a result of male factor defects. Of these, nearly half again are idiopathic, with the molecular underpinnings of infertility undefined. Using global and germ-cell specific knockout mouse models of RNA editing enzymes, this proposal will test the hypothesis that germ cell RNA editing results in changes to the germ cell proteome and is fundamental for male fertility. Aim 1 is to determine whether global loss of the previously described putative RNA editing regulator changes the frequency and/or site distribution of RNA editing in the testis. Aim 2 will establish if there is a requirement for a key RNA editing enzyme in germ cell development and fertility and determine if germ-cell specific loss of the enzyme alters testicular RNA editing. Lastly, Aim 3 will determine whether RNA editing in mRNA coding regions impacts the protein coding capacity or translation of edited targets by mass spectrometry detection of peptides coded for by edited transcripts and the polysomal association of edited transcripts relative to their unedited counterparts. The knowledge gained from these studies represents a substantial contribution to the fields of male reproduction and RNA editing and may provide insight into the mechanisms of idiopathic male infertility.