Interplay between transcription and translation

 描述(由申请人提供)：非常精确的细胞增殖控制对于正常的动物发育是必不可少的，而细胞分裂的放松管制是癌症的标志。E2F转录因子是调节哺乳动物分裂周期的机制的组成部分。E2F的作用在整个进化过程中一直是保守的。E2F协调细胞分裂和增殖所需的数百个基因的转录。自E2F首次被发现以来的二十年里，人们一直认为依赖于E2F的转录的激活必然导致编码蛋白合成的增加。我最近发现了另一层转录后调控的证据，它广泛地作用于E2F调控的相当一部分转录本，并抑制了翻译 这些信使核糖核酸。果蝇遗传学研究表明，两种RNA结合蛋白，Pumilio和Nanos，在体内功能上抑制E2F活性。分子研究证实，在果蝇和人类中，E2F/Rb和Pumilio/Nanos蛋白的调控密切相关，编码激活因子E2F的mRNAs通过Pres被抑制，Nanos和Pumilio基因的转录被E2F和Rb家族成员抑制。对这些联系的一种解释出现在“经济学”研究中。我们最近对与Rb突变相关的小鼠肺组织的转录和蛋白质组变化的比较表明，尽管Rb突变后E2F靶基因的转录显著增加，但这些基因中很少有蛋白质水平的变化。值得注意的是，对Rb突变组织中转录上调的mRNAs中转录后基序的无偏见分析发现，前基序是最显著的浓缩。此外，对视网膜母细胞瘤中上调的mRNA序列的分析表明，50%的E2F调节基因中的mRNAs含有假定的前基序。这一提议将检验这样一个假设，即Pumilio和Nanos蛋白提供了一层转录后调控，作用于广泛的E2F诱导的转录本。我将确定哪些E2F诱导的转录本在受到调控时被以这种方式抑制，并将调查这种调控对蛋白质水平的影响。放松调控的E2F活动导致了许多人类癌症的扩散。我将发现由Pumilio和Nanos介导的控制是否缓冲了RB突变细胞对E2F依赖的转录的增加，并将确定这个装置在癌细胞中是保留还是丢失。总的来说，这些实验将定义一种新的调控机制，限制E2F转位基因的表达。

 DESCRIPTION (provided by applicant): A very precise control of cell proliferation is essential for normal animal development and the deregulation of cell division is a hallmark of cancer. The E2F transcription factors are integral components of the machinery regulating the mammalian division cycle. The role of E2F has been conserved throughout evolution. E2F co- ordinates the transcription of hundreds of genes that are needed for cells to divide and proliferate. In the two decades since E2F was first identified it has been assumed that activation of E2F-dependent transcription inevitably results in the increased synthesis of the encoded proteins. I have recently uncovered evidence of an additional layer of post-transcriptional regulation that acts broadly on a significant fraction of E2F-regulated transcripts, and that suppresses the translation of these mRNAs. Drosophila genetic studies show that two RNA-binding proteins, Pumilio and Nanos, functionally suppress E2F activity in vivo. Molecular studies have confirmed that the regulation of E2F/RB and Pumilio/Nanos proteins are intimately interconnected in both flies and humans, with mRNAs encoding activator E2Fs being suppressed via Pumilio response elements (PREs), and the transcription of Nanos and Pumilio genes being suppressed by E2F and RB family members. An explanation for these connections has emerged from "omic" studies. Our recent comparison of the transcriptional and proteomic changes associated with RB- mutation in mouse lung tissue revealed that although the transcription of E2F target genes were dramatically increased upon RB mutation, very few of these genes displayed protein level changes. Strikingly, an unbiased analysis of the post-transcriptional motifs within mRNAs that are transcriptionally upregulated in RB mutant tissues, without a similar increase in protein levels, found that PRE-motifs are the most significantly enriched. Furthermore analysis of mRNA sequences upregulated in retinoblastoma tumors shows that mRNAs from >50% of E2F-regulated genes contain putative PRE motifs. This proposal will test the hypothesis that Pumilio and Nanos proteins provide a tier of post- transcriptional regulation that acts on a broad swath of E2F-induced transcripts. I will determine which E2F- induced transcripts are suppressed in this manner, when they are regulated, and will investigate the impact of this regulation on protein levels. Deregulated E2F activity drives the proliferation of many human cancers. I will discover whether the control mediated by Pumilio and Nanos buffers RB mutant cells from increases in E2F-dependent transcription, and I will determine whether this apparatus is preserved or lost in cancer cells. Collectively these experiments will define a novel regulatory mechanism that limits the expression of E2F- transcibed genes.