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修饰的调节中发挥关键作用；这些过程对细胞功能有广泛的影响。RNA结合蛋白(RBP)通过改变靶RNA的组成、稳定性或蛋白质结合来调节RNA的功能。虽然已经观察到了细胞特异性的核RNA调控，但其潜在的机制和功能后果仍不清楚。本研究的目的是确定两个雄性生殖细胞特异性限制性商业惯例(ADAD1和ADAD2)如何调节核RNA的生物发生，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Adad1模型的高通量RNA测序(RNA-seq)分析表明，尽管基因表达或异构体丰度几乎没有变化，但一类核RNAs(小核仁RNA-snoRNAs)的表达发生了实质性变化。SnoRNAs指导其他RNA，特别是核糖体RNA(RRNAs)的核苷酸修饰。不适当的rRNA修饰会导致核糖体功能障碍和异常翻译，这一过程在雄性生殖细胞中受到严格控制。这与ADAD1的核定位相结合，表明ADAD1在snoRNA的生物发生或调控中发挥着关键作用，与其密切相关的同系物AD2可能也是如此。这项拟议的研究将分两个阶段进行，指导阶段(I)和独立阶段(II)，并将检验ADS通过调节雄性生殖细胞中snoRNAs的生物发生来影响翻译的假设。为了解决这一假设，本研究将：1)建立Adad1突变对rRNA修饰和翻译的影响(阶段I)；2)证明ADAD1和2是否与snoRNAs或snoRNA调节蛋白相关(阶段II)；以及3)结合生化分析、RNA-SEQ和新的遗传模型确定Adad1或2对snoRNA丰度和生殖细胞发育的影响(阶段I)。最终，这项工作将确定ADADs、snoRNAs和生殖细胞翻译之间的联系，从而了解核RNAs影响男性生育能力的机制。

 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.