RNA uridylation as a protective mechanism in the germline

生育需要形成和维持健康的生殖细胞。指定生殖细胞前体 在早期胚胎中，随后增殖，经历减数分裂，并分化为精子或卵母细胞。 在整个发育过程中，保护机制确保生殖细胞抵抗外部影响， 可能会改变它的命运。该建议解决了RNA 3'尿苷化在调节基因表达中的作用， 以保护生殖细胞的命运并确保健康配子的发育。3'尿苷化是一种后 转录修饰对RNA稳定性和功能具有上下文依赖性影响。聚（U） 聚合酶是将尿苷残基添加到RNA分子的3'末端的核苷酸转移酶。我们发现 模型线虫C.线虫，临界效应生殖系 发展特别地，PUP-1和PUP-2的表达一起防止了PUP-1和PUP-2的异常表达。 体细胞基因，维持生殖细胞活力，并确保配子形成。哺乳动物直系同源物，TUT 4 ZCCHC 11和TUT 7（ZCCHC 6）对于种系和早期胚胎发育同样是必需的。 引人注目的是，在PUP-1和PUP-2缺失的情况下，我们发现PUP-3和PUP-4的表达有助于PUP-1和PUP-2的表达。 这些生殖缺陷。然而，PUP-1和PUP-2修饰小RNA的子集，TUT 4和TUT 7也是如此。 不知道PUP-3和PUP-4是否具有这种活性。TUT 4和TUT 7也修饰mRNA，但它们不是 已知C. Elegans PUP具有这种活性。我们有一个正在进行的项目，以确定PUP-1和PUP-2 目标的在这项提案中，我们将进行遗传，蛋白质表达和RNA测序研究，以测试 关于pup-3和pup-4相对于彼此和pup-4的发育作用的替代假设， 1和pup-2（目标1）。我们将进行RNA测序实验以鉴定尿苷酸化的小RNA， pup-3和pup-4单突变株和多突变株中的mRNA，包括pup-3;pup-1/-2和pup-4;pup-1/-2 三重突变体（Aim 2）。这些信息将使我们能够识别PUP-3的独特和共同目标， PUP-4活性和尿苷酸化的损失与RNA丰度的变化相关。我们假设 PUP-3和PUP-4活性的靶标不同于PUP-1和PUP-2活性的靶标。我们将测试 通过将此处获得的RNA测序数据与pup-1和pup-2 RNA数据集进行比较， 我们现在正在生成（目标2.C）。我们的中心假设是PUP活性调节了 小RNA和mRNA，以防止生殖细胞身份的丧失，以及确保种系活力和 生产健康的配子。

Fertility requires the formation and maintenance of a healthy germline. Germ cell precursors are specified in the early embryo and later proliferate, undergo meiosis, and differentiate as sperm or oocytes. Throughout development, protective mechanisms ensure that the germline resists outside influences that might alter its fate. This proposal addresses the role of RNA 3’ uridylation in regulating gene expression in order to protect germ cell fate and ensure the development of healthy gametes. 3’ uridylation is a post- transcriptional modification with context-dependent effects on RNA stability and function. Poly(U) polymerases are nucleotidyl transferases that add uridine residues to the 3’ end of RNA molecules. We find that all four poly(U) polymerase proteins in the model nematode, C. elegans, critically effect germline development. In particular, expression of PUP-1 and PUP-2 together prevents aberrant expression of somatic genes, maintains germ cell viability, and ensures gamete formation. Mammalian orthologs, TUT4 (ZCCHC11) and TUT7 (ZCCHC6), are likewise essential for germline and early embryonic development. Strikingly, in the absence of PUP-1 and PUP-2, we find that expression of PUP-3 and PUP-4 contribute to these germline defects. PUP-1 and PUP-2 modify subsets of small RNAs, as do TUT4 and TUT7, however it is unknown if PUP-3 and PUP-4 have this activity. TUT4 and TUT7 also modify mRNA, however it is not known which C. elegans PUPs have this activity. We have an ongoing project to identify PUP-1 and PUP-2 targets. In this proposal, we will perform genetic, protein expression, and RNA sequencing studies to test alternative hypotheses about the developmental roles of pup-3 and pup-4 relative to each other and to pup- 1 and pup-2 (Aim 1). We will perform RNA sequencing experiments to identify uridylated small RNAs and mRNAs in pup-3 and pup-4 single and multiple-mutant strains, including pup-3;pup-1/-2 and pup-4;pup-1/-2 triple mutants (Aim 2). This information will allow us to identify unique and common targets of PUP-3 and PUP-4 activity and correlate loss of uridylation with changes in RNA abundance. We hypothesize that the targets of PUP-3 and PUP-4 activity are different from the targets of PUP-1 and PUP-2 activity. We will test this hypothesis by comparing RNA-sequencing data obtained here with the pup-1 and pup-2 RNA data sets we are now generating (Aim 2.C). Our central hypothesis is that PUP activity modulates the abundance of small RNAs and mRNAs to prevent the loss of germ cell identity, as well as to ensure germline viability and production of healthy gametes.