Investigating the molecular functions and mechanisms of RNA-binding proteins crucial for gametogenesis

在试图怀孕的夫妇中，10 - 15%会受到不孕症的影响，这可能是由以下原因引起的： 配子发生受损通过RNA结合蛋白（RBP）进行有效的转录后基因调控 对于正常的配子发生至关重要，RBPs的突变会削弱这种调节， 人类的不孕症例如，RBP的无精子症（DAZ）家族对于以下是必需的： 人类生育能力，因为在精子发生过程中，它们通过以下方式调节一部分mRNA的翻译： 结合其3 ′ UTR;其分子作用机制只有在其 果蝇的直系同源物被鉴定并进行了遗传学研究。然而，所有限制性商业惯例， 目前还不清楚配子发生所必需的。为了充分理解复杂的转录后 在配子发生过程中的基因调控，关键是要确定新的RBP是重要的， 过程，并阐明其分子功能和作用机制。 我们最近发现了三个以前未表征的RBP（MARF 1，Tanenashi和 CG44249），是果蝇生育力所必需的。我们的数据表明，MARF 1，这是 在卵母细胞中表达，并且是雌性生育力所必需的，调节mRNA poly-A尾长， 卵子发生Tanenashi在精母细胞中表达，是男性生育所必需的， 在促进含有巨碱基大小的雄性育性因子基因的表达和剪接中的作用 精子发生过程中Y染色体上具有高度重复DNA序列的内含子。CG44249， 在卵巢中表达，是女性生育所必需的，可能在剪接调节中起作用。 在卵子发生过程中。我们还表明，第四个RBP（Loqs-PB），这是重要的女性生育能力， 和生殖系干细胞的维持，调节卵巢中microRNA的长度。这些 四种RBP是从苍蝇到人类的保守性。 利用果蝇，我现在建议进一步研究这四种RBP，并确定它们的 精确的分子功能和配子发生的作用机制。通过调查变异苍蝇 在体内和体外的蛋白质，我们将测试我们的假设，即：（i）这些RBP各自结合特异性靶点 通过识别特定的RNA序列和/或结构基序，和（ii）从而促进或抑制 在特定的分子步骤中对结合的RNA进行加工， 配子发生过程中靶基因的表达。 这些研究将推进我们对后- 基因表达的转录调节，特别是通过调节mRNA poly-A尾长， mRNA剪接和microRNA长度，在配子发生过程中，提供了改善的潜力， 诊断和治疗人类不育症。

Among couples trying to conceive, 10-15% will be affected by infertility, which can be caused by impaired gametogenesis. Effective post-transcriptional gene regulation by RNA-binding proteins (RBPs) is crucial for normal gametogenesis, with mutations in RBPs impairing such regulation and causing infertility in humans. For example, the Deleted in Azoospermia (DAZ) family of RBPs are essential for human fertility, because during spermatogenesis they regulate translation of a subset of mRNAs by binding to their 3′ UTRs; their molecular mechanism of action was understood only after their Drosophila ortholog was identified and studied genetically. However, the full repertoire of RBPs that are essential for gametogenesis is currently unknown. To fully understand the complex post-transcriptional gene regulation during gametogenesis, it is critical to identify novel RBPs that are important for these processes and elucidate their molecular functions and mechanisms of action. We have recently identified three previously uncharacterized RBPs (MARF1, Tanenashi, and CG44249) that are essential for fertility in Drosophila. Our data suggest that MARF1, which is expressed in oocytes and is required for female fertility, regulates mRNA poly-A tail length during oogenesis. Tanenashi, which is expressed in spermatocytes and is required for male fertility, plays roles in promoting the expression and splicing of male fertility factor genes containing mega-base-sized introns with highly repetitive DNA sequence on the Y chromosome during spermatogenesis. CG44249, which is expressed in ovaries and is required for female fertility, likely plays a role in splicing regulation during oogenesis. We also showed that a fourth RBP (Loqs-PB), which is important for female fertility and germline stem cell maintenance in ovaries, regulates the length of microRNAs in ovaries. These four RBPs are conserved from flies to humans. Using Drosophila, I now propose to further investigate these four RBPs, and determine their precise molecular functions and mechanisms of action in gametogenesis. By investigating mutant flies in vivo and the proteins in vitro, we will test our hypothesis that: (i) these RBPs each bind specific target RNAs by recognizing specific RNA sequence and/or structure motifs, and (ii) thereby promote or inhibit the processing of bound RNAs at specific molecular steps, allowing highly regulated spatiotemporal expression of targeted genes during gametogenesis. These studies will advance our fundamental knowledge of the molecular mechanisms of post- transcriptional regulation of gene expression, especially by regulating the mRNA poly-A tail length, mRNA splicing, and microRNA length, during gametogenesis, providing potential for improved diagnosis and treatment of human infertility.