Male Germ Cell RNA Binding Proteins and Nuclear RNAs in Male Fertility

 描述（由申请人提供）：核RNA在转录、剪接和RNA修饰的调控中发挥关键作用;这些过程对细胞功能具有广泛影响。RNA结合蛋白（RBP）通过改变靶RNA的组成、稳定性或蛋白质缔合来调节RNA功能。虽然已经观察到细胞特异性的核RNA调节，但其潜在机制和功能后果仍不清楚。本研究的目的是确定两种雄性生殖细胞特异性RBP（ADAD 1和ADAD 2）如何调节核RNA生物合成以及这种调节对精子发生的功能影响。这项研究建立在我的NIH博士后奖学金支持的研究基础上。对雄性不育突变体Adad 1模型的高通量RNA测序（RNA-seq）分析表明，尽管基因表达或同种型丰度几乎没有变化，但一类核RNA（小核仁RNA-snoRNA）的表达发生了实质性变化。snoRNA引导其他RNA，特别是核糖体RNA（rRNA）中的核苷酸修饰。不正确的rRNA修饰导致核糖体功能障碍和异常翻译，这是一个在雄性生殖细胞中受到严格控制的过程。这与ADAD 1核定位相结合，表明ADAD 1在snoRNA生物发生或调节中起关键作用，其密切相关的同源物ADAD 2也是如此。拟议的研究将分两个阶段进行，指导阶段（I）和独立阶段（II），并将测试ADAD通过调节雄性生殖细胞中snoRNA的生物发生来影响翻译的假设。为了验证这一假设，本研究将：1）确定Adad 1突变对rRNA修饰和翻译的影响（I期）; 2）证明ADAD 1和2是否与snoRNA或snoRNA调节蛋白相关（第二阶段）;和3）使用生物化学分析，RNA-seq，和新的遗传模型。最终，这项工作将确定ADAD，snoRNA和生殖细胞翻译之间的联系，从而了解核RNA影响男性生育力的机制。

 DESCRIPTION (provided by applicant): Nuclear RNAs play key roles in the regulation of transcription, splicing, and RNA modifications; processes that have widespread impacts on cellular function. RNA binding proteins (RBPs) modulate RNA function by altering the composition, stability, or protein association of target RNAs. Although cell-specific nuclear RNA regulation has been observed, the underlying mechanisms and functional consequences have remained unclear. The goal of this research is to determine how two male germ cell-specific RBPs (ADAD1 and ADAD2) regulate nuclear RNA biogenesis and the functional impact this regulation has on spermatogenesis. This research builds upon studies supported by my NIH postdoctoral fellowship. High-throughput RNA sequencing (RNA- seq) analysis of a male infertile mutant Adad1 model demonstrated substantial changes in the expression of a class of nuclear RNAs (small nucleolar RNA - snoRNAs) in spite of virtually no changes in gene expression or isoform abundance. snoRNAs guide nucleotide modification in other RNAs, particularly ribosomal RNAs (rRNAs). Improper rRNA modification leads to ribosome malfunction and abnormal translation, a process that is tightly controlled in the male germ cell. This, combined with ADAD1 nuclear localization, suggests that ADAD1 plays a critical role in snoRNA biogenesis or regulation, as may its closely-related homologue ADAD2. The proposed research will occur in two phases, a mentored (I) and an independent phase (II) and will test the hypothesis that ADADs impact translation by regulating the biogenesis of snoRNAs in male germ cells. To address the hypothesis, this research will: 1) establish the impact of Adad1 mutation on rRNA modification and translation (phase I); 2) demonstrate whether ADAD1 and 2 associate with snoRNAs or snoRNA-regulating proteins (phase II); and 3) determine the impact of Adad1 or 2 ablation on snoRNA abundance and germ cell development (phase I) using a combination of biochemical analysis, RNA-seq, and novel genetic models. Ultimately, this work will define the connection between ADADs, snoRNAs and germ cell translation thus informing on the mechanisms by which nuclear RNAs affect male fertility.