A New Paradigm for Cell Size Control by the RB Tumor Suppressor Pathway

描述(由申请人提供)：大小控制是细胞的一项基本属性，涉及对生长和分裂的协调调节，这一过程在癌症等人类疾病中经常出现功能障碍。这项研究的长期目标是建立管理规模控制的机械性原则。我们已经开发了一种独特的信息模型来调节细胞大小，单细胞藻类莱茵衣藻。利用衣藻，我们已经确定了大小检查点途径的关键成分，其行为表明了一种将细胞大小与细胞周期激活相结合的机制。该检查点的每个基因识别组件都与人类对应，包括视网膜母细胞瘤(RB)肿瘤抑制途径的蛋白、使RB磷酸化的细胞周期蛋白依赖激酶CDKG1和RNA结合蛋白TNY1。我们假设，CDKG1的活性由TNY1控制，作为连接细胞大小和细胞周期进入的一种手段，这两种活性的平衡通过开启和关闭RB途径来控制细胞周期。这一建议的发现将提供有关细胞生长和分裂控制机制的基础知识，这些机制与理解人类的类似过程有关。本研究的具体目的如下：1.研究CDKG1作为SIZER蛋白的作用：我们将确定CDKG1在细胞周期中的丰度和活性是如何被控制的；ii)确定CDKG1底物Rb相关蛋白MAT3上的CDKG1磷酸化位点；iii)利用条件等位基因来确定CDKG1在细胞周期中的时间需求。目的2.确定TNY1抑制细胞周期的机制：TNY1在每个细胞周期产生一次，然后随着细胞在G1期的生长而稀释。我们将测试TNY1作为对抗CDKG1活性的细胞大小的阈值调节因子的作用。我们将确定TNY1是否通过结合其mRNA来调节CDKG1；ii)测试TNY1的剂量效应；iii)识别内源性TNY1 RNA靶点；以及iv)确定靶向TNY1是否带来编码蛋白在TNY1和/或细胞周期控制下的表达。目的3.通过抑制子筛选发现新的大小途径基因：Tny1缺失突变体和CDKG1过表达(CDKG1-OE)系都具有小细胞表型，为识别大小控制的相互作用成分提供了敏感的遗传背景。I)我们将在前向筛选中使用这些线来寻找其小细胞表型的抑制因子。这些抑制子被预测为编码靶蛋白或相互作用的蛋白。Ii)我们将使用二级筛选来对大小途径中的突变体进行排序，并确定用于进一步研究和克隆的候选基因。

DESCRIPTION (provided by applicant): Size control is a fundamental property of cells that involves coordinated regulation of growth and division, processes that are frequently dysfunctional in human diseases such as cancer. The long-term goal of this research is to establish the mechanistic principles that govern size control. We have exploited a uniquely informative model for cell-size regulation, the unicellular alga Chlamydomonas reinhardtii. Using Chlamydomonas we have identified key components of a size checkpoint pathway whose behavior suggests a mechanism for coupling cell size to cell cycle activation. The genetically identified components of this checkpoint each have human counterparts and include proteins of the retinoblastoma (RB) tumor suppressor pathway, a cyclin dependent kinase, CDKG1, that phosphorylates RB, and an RNA binding protein, TNY1. We hypothesize that the activity of the kinase, CDKG1, is held in check by TNY1 as a means of coupling cell size to cell cycle entry, and that the balance of these two activities controls the cell cycle by switching on and off the RB pathway. The findings from this Proposal will provide fundamental knowledge regarding mechanisms of cell growth and division control that are relevant for understanding similar processes in humans. The Specific Aims of this Proposal are as follows: Aim 1. Investigate the role of CDKG1 as sizer protein: We will i) determine how CDKG1 abundance and activity are controlled during the cell cycle; ii) identify and characterize CDKG1 phosphorylation sites on its substrate, the RB related protein MAT3; iii) determine the temporal requirement for CDKG1 in the cell cycle using conditional alleles. Aim 2. Determine the mechanism by which TNY1 represses the cell cycle: TNY1 is produced once per cell cycle and then becomes diluted as cells grow during G1. We will test the role of TNY1 as a threshold regulator of cell size that opposes the activity of CDKG1. We will i) establish whether TNY1 regulates CDKG1 through binding its mRNA; ii) test dosage effects of TNY1; iii) identify endogenous TNY1 RNA targets; and iv) establish whether targeting TNY1 to a mRNA brings expression of the encoded protein under control of TNY1 and/or the cell cycle. Aim 3. Identify new size pathway genes through suppressor screens: tny1 null mutants and CDKG1 over- expressing (CDKG1-OE) lines both have small-cell phenotypes that provide a sensitized genetic background in which to identify interacting components of size control. i) We will use these lines in forward screens to find suppressors of their small-cell phenotypes. These suppressors are predicted to encode either targets or interacting proteins. ii) We will use secondary screens to order the mutants in the size pathway and to identify candidates for further investigation and cloning.