Post-transcriptional regulation of development

生产有生育能力的精子需要表达精子发育不同阶段所需的1000多种蛋白质。例如，一些蛋白质在精子发育的早期起作用，以确保精子包含正确数量的染色体。其他蛋白质在精子发育后期起作用，形成鞭状尾巴，在受精期间将精子推向卵子。尽管最近在理解精子细胞如何发育方面取得了进展，但人们对蛋白质是如何在正确的地点和时间形成可育精子的知之甚少。我们最近发现，一种名为eIF4E-3的因子控制着果蝇精子发育过程中的蛋白质合成。EIF4E-3是调节蛋白质合成起始的保守蛋白质家族成员。在缺乏eIF4E-3的情况下，一些精子蛋白的表达水平低于正常水平，干扰细胞分裂和精子发育，并导致男性不育。出乎意料的是，典型的eIF4E(果蝇中的eIF4E-1)的敲除类似于eIF4E-3的丢失，这表明eIF4E-3和eIF4E-1可能调节类似的蛋白质。然而，eIF4E-3缺少重要的氨基酸，这表明它的调控可能与eIF4E-1不同。此外，果蝇还会产生另外三种睾丸特异的eIF4E蛋白：eIF4E-4、-5和-7。拟议的实验将结合细胞生物学、遗传学、分子生物学和生物化学来发现eIF4E-3的作用和调控，确定其他eIF4E的作用，并确定这些eIF4E在精子发育过程中调节的靶基因。*我们的实验首次系统地描述了果蝇雄性生殖系的翻译调控机制，将集中在以下具体目标上：*1.分析eIF4E-3在精子发生中的作用和调控。*我们将使用荧光显微镜来研究eIF4E-3突变体的缺陷，并将它们与eIF4E-1的缺失进行比较。我们将对eIF4E-4、-5和-7进行基因敲除，并研究它们在精子发生中的作用。我们将使用遗传杂交来测试eIF4E-3和eIF4E-1是否具有重叠或独特的作用，并进行挽救实验来测试eIF4E-3中缺失的氨基酸的重要性。*2.研究睾丸特异的eIF4F复合体及其靶点。*我们将使用免疫共沉淀(co-IP)来鉴定睾丸中存在的蛋白质合成复合体。我们将使用质谱仪来测试eIF4E-3的序列变化是否会影响其调控。我们将进行RNA共IP，以确定优先与在睾丸中表达的五个eIF4E中的每一个相关的mRNAs集。*意义：我们的实验将揭示翻译控制在果蝇雄性生殖细胞发育中的作用和机制，并揭示动物发育过程中特定细胞分化关键步骤的翻译控制调控的广泛原理。

Production of fertile sperm requires expression of more than 1000 proteins that are needed at different stages of sperm development. For example, some proteins act early in sperm development to ensure the sperm contains the correct number of chromosomes. Other proteins act later in sperm development to form the whip-like tail that propels the sperm towards the egg during fertilization. Despite recent progress in understanding how sperm cells develop, little is known about how proteins are made at the right place and time to form fertile sperm. We recently discovered that a factor called eIF4E-3 controls protein synthesis during sperm development in fruit flies. eIF4E-3 is a member of a conserved family of proteins that regulates initiation of protein synthesis. In the absence of eIF4E-3, some sperm proteins are expressed at lower than normal levels, interfering with cell division and sperm development and causing male sterility. Unexpectedly, knockdown of the canonical eIF4E (eIF4E-1 in flies) resembles loss of eIF4E-3, suggesting eIF4E-3 and eIF4E-1 may regulate similar proteins. However, eIF4E-3 is missing important amino acids, suggesting it may be regulated differently from eIF4E-1. In addition, fruit flies make three other testis-specific eIF4E proteins, eIF4E-4, -5 and -7. The proposed experiments will use a combination of cell biology, genetics, molecular biology and biochemistry to discover the role and regulation of eIF4E-3, determine the roles of the other eIF4Es, and identify target genes regulated by these eIF4Es during sperm development. *** Our experiments, which will be the first to systematically characterize mechanisms of translational regulation in the fruit fly male germline, will focus on the following specific aims:*******1. Analyze the role and regulation of eIF4E-3 in spermatogenesis. ***We will use fluorescence microscopy to study the defects in eIF4E-3 mutants, and to compare them with loss of eIF4E-1. We will engineer knockouts of eIF4E-4, -5 and -7 and study their roles in spermatogenesis. We will use genetic crosses to test whether eIF4E-3 and eIF4E-1 have overlapping or unique roles, and perform rescue experiments to test the significance of the amino acids that are missing in eIF4E-3.******2. Investigate testis-specific eIF4F complexes and their targets. ***We will use co-immunoprecipitation (co-IP) to identify the protein synthesis complexes present in testes. We will use mass spectrometry to test whether sequence changes in eIF4E-3 affect its regulation. We will carry out RNA co-IPs to identify sets of mRNAs that preferentially associate with each of the five eIF4Es expressed in testes.******Significance: Our experiments will reveal the role and mechanism of translational control in fruit fly male germ cell development and will uncover broad principles underlying translational control regulation of key steps in differentiation of specialized cells during animal development.**