Interplay between transcription and translation

 描述（由申请人提供）：细胞增殖的非常精确的控制对于正常动物发育是必不可少的，细胞分裂的失调是癌症的标志。E2 F转录因子是调节哺乳动物分裂周期的机制的组成部分。E2 F的作用在整个进化过程中一直是保守的。E2 F协调细胞分裂和增殖所需的数百个基因的转录。自E2 F首次被鉴定以来的二十年中，人们一直认为E2 F依赖性转录的激活不可避免地导致编码蛋白质的合成增加。我最近发现了转录后调节的另一层的证据，它广泛地作用于E2 F调节的转录物的很大一部分，并且抑制翻译。 这些mRNA。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E2 F活性。分子研究已经证实，E2 F/RB和Pumilio/Nanos蛋白的调节在果蝇和人类中密切相关，编码激活剂E2 F的mRNA通过Pumilio反应元件（PRE）被抑制，Nanos和Pumilio基因的转录被E2 F和RB家族成员抑制。对这些联系的解释已经出现在“组学”研究中。我们最近比较了小鼠肺组织中与RB突变相关的转录和蛋白质组学变化，结果显示，尽管RB突变后E2 F靶基因的转录显著增加，但这些基因中很少显示蛋白质水平的变化。引人注目的是，在RB突变组织中转录上调的mRNA内的转录后基序的无偏分析，没有类似的蛋白质水平的增加，发现PRE基序是最显著的富集。此外，对视网膜母细胞瘤肿瘤中上调的mRNA序列的分析表明，来自>50%的E2 F调节基因的mRNA含有推定的PRE基序。该提议将测试Pumilio和Nanos蛋白提供一层转录后调节的假设，该转录后调节作用于广泛的E2 F诱导的转录物。我将确定哪些E2 F诱导的转录本以这种方式被抑制，当它们被调节时，并将研究这种调节对蛋白质水平的影响。E2 F活性失调驱动许多人类癌症的增殖。我将发现Pumilio和Nanos介导的控制是否缓冲了RB突变细胞的E2 F依赖性转录的增加，我将确定这种装置在癌细胞中是保留还是丢失。总的来说，这些实验将确定一种新的调节机制，限制E2 F-转基因的表达。

 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.