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

研究对配子发生至关重要的 RNA 结合蛋白的分子功能和机制

• 批准号: 10630355
• 负责人: Ryuya Fukunaga
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630355
• 关键词: 3' Untranslated Regions; Address; Affect; Binding; Cell Maintenance; Complex; Couples; DNA Sequence; Data; Diagnosis; Drosophila genus; F Factor; Family; Fertility; Gametogenesis; Gene Expression; Gene Expression Regulation; Genes; Human; Impairment; In Vitro; Infertility; Introns; Knowledge; Length; Messenger RNA; MicroRNAs; Molecular; Molecular Mechanisms of Action; Mutation; Oocytes; Oogenesis; Orthologous Gene; Ovary; Play; Poly(A) Tail; Post-Transcriptional Regulation; Process; Production; Proteins; RNA; RNA Binding; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Role; Science; Spermatocytes; Spermatogenesis; Structure; Testing; Translations; Y Chromosome; egg; female fertility; fly; germline stem cells; improved; in vivo; infertility treatment; male fertility; mutant; novel; polyadenylated messenger RNA; programs; spatiotemporal; sperm cell; trying to conceive

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.

在试图受孕的夫妇中，有不育会影响10-15％ 配子发生受损。 RNA结合蛋白（RBP）调节有效的转录后基因调节 对于正常的配子发生至关重要，RBP中的突变会损害这种调节并引起 人类不孕。例如，在RBP的Azoospermia（DAZ）家族中已删除的该删除对于 人类的生育能力，因为在精子发生过程中，它们调节了mRNA子集的翻译 与他们的3'UTR结合；他们的分子作用机理只有在他们的分子机制才能理解 果蝇直系同源物被鉴定并进行了研究。但是，RBP的完整曲目 对于配子发生的必不可少的目前尚不清楚。充分了解复杂的转录后 基因调节在配子发生过程中，至关重要的是识别对这些重要的新型RBP 过程并阐明其分子功能和作用机制。 我们最近确定了三个以前未表征的RBP（Marf1，Tanenashi和 CG44249）对于果蝇中的生育至关重要。我们的数据表明MARF1，那是 在卵母细胞中表达，是女性生育需要的，调节mrna poly-a尾巴长度 卵子发生。用精子细胞表达的Tanenashi，是男性生育的必需品 在促进含有巨型基尺寸的男性生育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， 这是在卵巢中表达的，是女性生育需要的，可能在剪接调节中起作用 在卵子发生期间。我们还表明了第四rbp（LOQS-PB），这对于女性生育很重要 和种系干细胞维持卵巢，调节卵巢中的microRNA的长度。这些 从苍蝇到人类，有四个RBP是保守的。 使用果蝇，我现在建议进一步研究这四个RBP，并确定它们 精确的分子函数和配子发生中的作用机制。通过研究突变蝇 体内和体外蛋白 通过识别特定的RNA序列和/或结构基序，RNA，（ii）从而促进或抑制 在特定分子步骤上处理结合的RNA，允许高度调节的时空 在配子发生过程中靶向基因的表达。 这些研究将提高我们对后分子机制的基本知识 基因表达的转录调节，尤其是通过控制mRNA poly-a尾长的长度， 配子发生过程中的mRNA剪接和microRNA长度，为改进的潜力提供了 人类不育的诊断和治疗。