RNA Editing in the Neonatal and Adult Testis

描述（由申请人提供）：RNA编辑，即RNA物种内核苷酸的修饰，广泛存在于哺乳动物中。RNA编辑具有改变蛋白质编码转录物的编码能力以及修饰靶向转录物的转录后调节的能力。高通量RNA测序的最新进展已经大大增加了用于RNA编辑的已知靶标的数量以及已经观察到RNA编辑的组织的数量。不幸的是，RNA编辑的目标和结果对于许多系统来说都是不清楚的。这项工作的目标是确定RNA编辑在男性生育力中的作用，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，假定的RNA编辑调节因子的全局消融导致男性不育。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RNA编辑是否是导致某些男性不育的原因。每七对夫妇中就有一对患有不孕症， 男性因素缺陷导致的病例。其中，又有近一半是特发性的，不孕症的分子基础尚不清楚。使用RNA编辑酶的全局和生殖细胞特异性敲除小鼠模型，该提案将测试生殖细胞RNA编辑导致生殖细胞蛋白质组变化并且对男性生育力至关重要的假设。目的1是确定先前描述的假定RNA编辑调节子的全局丢失是否改变睾丸中RNA编辑的频率和/或位点分布。目标2将确定生殖细胞发育和生育力中是否需要关键的RNA编辑酶，并确定生殖细胞特异性酶的丢失是否会改变睾丸RNA编辑。最后，目标3将确定mRNA编码区的RNA编辑是否影响蛋白质编码能力或编辑靶点的翻译，方法是通过质谱检测编辑转录物编码的肽以及编辑转录物相对于其未编辑对应物的多聚体缔合。从这些研究中获得的知识代表了对男性生殖和RNA编辑领域的重大贡献，并可能为特发性男性不育的机制提供见解。

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.