Regulation of Spermatogenesis by X-linked miRNAs

描述（由申请方提供）：精子发生期间减数分裂性染色体失活（MSCI）的特征是粗线期中期至晚期精母细胞中X和Y染色体上的基因转录沉默。MSCI被认为是由未配对DNA的减数分裂沉默引起的，因为X和Y染色体在整个第一次减数分裂前期大部分保持未配对。因此，与女性胚胎或体细胞中的X染色体失活不同，其中25-30%的X连锁结构基因已被报道逃脱失活，先前没有报道初级精母细胞中逃脱MSCI沉默效应的基因。然而，我们最近发现，许多X-连锁microRNA（miRNA）的转录和加工粗线期精母细胞。这种前所未有的从MSCI中逃逸的现象表明，这些miRNAs在精子发生的这个阶段参与了一种或多种关键功能。我们的初步发现证实了这一点，即携带高百分比的X连锁miRNA基因主要簇敲除细胞的嵌合小鼠在精子发生的减数分裂阶段显示出生育缺陷，表现为阻滞。这是重要的，因为尽管最近的报道描述了大量的miRNA和其他小的非编码RNA在精子发生过程中的表达，但基本上对这些中的任何一个的功能都一无所知。在本申请中，我们首先提出研究这些X连锁的miRNA基因逃避MSCI（Aim 1）的分子机制。然后，我们提出测试关于这些miRNA的功能的两个假设（它们不是相互排斥的），包括它们作为在减数分裂期间合成但直到减数分裂后期才翻译的常染色体mRNA的转录后调节剂的作用（Aim 2），以及它们在调节MSCI本身的过程中的作用（Aim 3）。这项研究非常新颖，因为它旨在揭示一种前所未有的逃离MSCI的机制，并确定在精子发生期间经历这种逃离的X连锁miRNA的实际功能。

DESCRIPTION (provided by applicant): Meiotic sex chromosome inactivation (MSCI) during spermatogenesis is characterized by transcriptional silencing of genes on both the X and Y chromosomes in mid to late pachytene spermatocytes. MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase. Thus, unlike X-chromosome inactivation in female embryonic or somatic cells, where 25-30% of X-linked structural genes have been reported to escape inactivation, there have been no previous reports of genes that escape the silencing effects of MSCI in primary spermatocytes. However, we recently discovered that many X-linked microRNAs (miRNAs) are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI suggests that these miRNAs participate in one or more critical functions at this stage of spermatogenesis. This is corroborated by our preliminary finding that chimeric mice carrying a high percentage of cells bearing a knockout of a major cluster of X-linked miRNA genes display a fertility defect manifest as a block during the meiotic phase of spermatogenesis. This is significant because despite recent reports describing expression of an abundance of miRNAs and other small, non-coding RNAs during spermatogenesis, essentially nothing is known about the function of any of these. In this application, we first propose to investigate the molecular mechanism by which these X-linked miRNA genes escape MSCI (Aim 1). We then propose to test two hypotheses (which are not mutually exclusive) regarding function of these miRNAs, including their role as post- transcriptional regulators of autosomal mRNAs that are synthesized during meiosis but not translated until the postmeiotic period (Aim 2), and their role in regulating the process of MSCI itself (Aim 3). This study is highly novel in that it is designed to reveal an unprecedented mechanism of escape from MSCI, and to identify actual functions of those X-linked miRNAs that undergo this escape during spermatogenesis.