C. elegans RNA regulatory protein complexes that control germ cell fate

描述(申请人提供)：我的提案的长期目标是了解RNA-蛋白质复合体如何调节翻译以影响细胞命运。线虫成体生殖系提供了一个简单的模型来研究涉及影响生殖细胞(GSC)决定增殖或分化为配子的分子控制的问题。GSC命运的决定由FBF-1和FBF-2(统称为FBF)协调，它们是高度保守的PUF(Pumilio和FBF)蛋白家族中的冗余成员。FBF与分化所必需的特定信使RNA(MRNAs)结合，并在体外被证明与去烯化(CCF-1/非去烯化复合体)和多腺化(GLD-3/GLD-2细胞质聚(A)聚合酶)蛋白有关。在一个工作模型中，FBF与生殖系不同区域的特定Poly(A)尾部修饰蛋白相互作用，调节参与GSC命运决定的目标mRNAs的表达。下面的建议结合了遗传学、细胞生物学和生物化学的成熟技术，以及荧光显微镜的最新发展，以加深我们对FBF复合体的组装和空间调节的理解。我将以三个具体目标来测试我们的模型：1)我将确定可以在最小的体外蛋白质相互作用网络中与FBF组装的特定蛋白质组合。2)使用先进的荧光成像技术，我将可视化FBF与其结合伙伴相互作用的生殖线上的位置以及亚细胞位置。3)我将在体内测试FBF腺化修饰蛋白复合体是否影响FBF相关转录本的Poly(A)尾长。总之，我的建议将增加我们对RNA调节蛋白复合体如何控制多细胞有机体中的组织发育的总体理解。

DESCRIPTION (provided by applicant): The long-term goal of my proposal is to understand how RNA-protein complexes regulate translation to influence cell fate. The C. elegans adult germ line provides a simple model to study questions involving the molecular controls influencing a germ stem cell's (GSC's) decision to proliferate or differentiate into gametes. The GSC fate decision is coordinated by FBF-1 and FBF-2 (collectively referred to as FBF), redundant members of the highly conserved PUF (Pumilio and FBF) protein family. FBF binds to specific messenger RNAs (mRNAs) necessary for differentiation, and is shown in vitro to associate with deadenylating (CCF-1/Not deadenylation complex) and polyadenylating (GLD-3/GLD-2 cytoplasmic poly(A) polymerase) proteins. In a working model, FBF interacts with specific poly(A) tail modifying proteins in different regions of the germ line to regulate expression of target mRNAs involved in GSC fate decisions. The following proposal combines established techniques in genetics, cell biology, and biochemistry, with newer developments in fluorescent microscopy, to further our understanding of the assembly and spatial regulation of FBF complexes. I will test our model with three specific aims: 1) I will determine the specific protein combinations that can assemble with FBF in a minimal in vitro protein interaction network. 2) Using advanced fluorescent imaging techniques, I will visualize where in the germ line and what subcellular location FBF interacts with its binding partners. 3) I will test in vivo whether FBF adenylation-modifying protein complexes affect the poly(A) tail length of FBF- associated transcripts. Together, my proposal will increase our general understanding of how RNA regulatory protein complexes control tissue development in a multi-cellular organism.