A Novel Factor Involved in Translation of Histone mRNA and Subsets of PolyA mRNA

描述(申请人提供)：组蛋白mRNA水平与细胞周期协调调节，在S期开始积累，在S期结束时降解。这些依赖后生动物复制的组蛋白mRNAs是独一无二的，因为与其他mRNAs不同的是，它们不是多腺化的，而是以保守的茎环结构结束。这个独特的3‘端由茎环结合蛋白(SLBP)结合，它在组蛋白mRNA的加工、翻译和降解中发挥作用。我的研究重点是了解Marzluff实验室发现的一种结合SLBP的新型蛋白质的作用，这种蛋白质名为SLIP1。初步研究表明，SLIP1参与组蛋白mRNA的翻译，并可能参与部分聚(A)mRNA的翻译，是一种必需基因。这项提议的广泛的长期目标是确定SLIP1作为翻译因子的作用的分子细节，并确定除了组蛋白mRNA外，哪些细胞mRNAs还需要SLIP1进行翻译。为了将SLIP1作为一个翻译因子，我将使用分子和生化技术。利用非洲爪哇卵母细胞和哺乳动物细胞进行体内翻译分析，将确定对SLIP1介导的翻译至关重要的mRNA顺式元件。其他生化方法将被用来检查SLIP1与一般翻译因子和核糖体的结合。为了鉴定SLIP1结合蛋白，我将进行以SLIP1为诱饵的酵母双杂交筛选，以及SLIP1的免疫沉淀和质谱分析。识别的蛋白质将通过翻译实验进行验证，以确认SLIP1和识别的蛋白质之间的功能相互作用。受SLIP1翻译调控的细胞mRNAs将通过免疫沉淀SLIP1-RNP复合体，随后对沉淀的mRNAs进行微阵列分析来鉴定。微阵列分析将通过与迈克尔·惠特菲尔德博士的实验室(达特茅斯大学)合作完成，后者在微阵列分析方面拥有专业知识，之前曾与Marzluff实验室合作。通过这种方法鉴定的mRNAs将通过体内翻译试验进行测试，以确定SLIP1对其翻译的影响。组蛋白mRNA水平与细胞周期相协调，以确保在DNA合成过程中组蛋白含量丰富。导致组蛋白产生缺陷的突变可导致肿瘤发生、DNA损伤、细胞周期缺陷和细胞死亡。因此，了解调控组蛋白mRNA翻译的因素将有助于我们对癌症的理解。

DESCRIPTION (provided by applicant): Histone mRNA levels are regulated coordinately with the cell cycle, accumulating at the beginning of Sphase and being degraded at the end of S-phase. These metazoan replication-dependent histone mRNAs are unique, in that unlike other mRNAs, they are not polyadenylated, instead ending in a conserved stemloop structure. This unique 3' end is bound by the stemloop binding protein (SLBP), which plays a role in histone mRNA processing, translation, and degradation. My studies focus on understanding the role of a novel protein discovered in the Marzluff lab which binds SLBP, named SLIP1. Preliminary data has shown that SLIP1 is involved in histone mRNA translation and possibly in the translation of a subset of poly (A) mRNAs, and is an essential gene. The broad long-term objectives of this proposal are to define the molecular details of the role of SLIP1 as a translation factor and to determine what cellular mRNAs in addition to histone mRNAs require SLIP1 for translation. To characterize SLIP1 as a translation factor, I will employ molecular and biochemical techniques. In vivo translation assays using both Xenopus oocytes and mammalian cells will determine mRNA cis-elements important for SLIP1-mediated translation. Additional biochemical approaches will be used to examine SLIP1 binding to general translation factors and ribosomes. To identify SLIP1 binding proteins, I will perform both a yeast-two-hybrid screen using SLIP1 as bait, and immunoprecipitations of SLIP1 followed by mass spectrometry. Proteins identified will be validated by a translation assay to confirm a functional interaction between SLIP1 and proteins identified. Celluar mRNAs translationally regulated by SLIP1 will be identified through immunoprecipitation of SLIP1-RNP complexes, followed by microarray analysis of precipitated mRNAs. Microarray analysis will be accomplished through a collaboration with Dr. Michael Whitfield's lab (Dartmouth University), who have expertise with microarray analysis and have previously collaborated with the Marzluff lab. mRNAs identified through this approach will be tested by in vivo translation assays to determine the affect of SLIP1 on their translation. Histone mRNA levels are coordinated with the cell cycle to ensure that histone proteins are abundant during DNA synthesis. Mutations causing defects in histone protein production can lead to oncogenesis, DNA damage, cell cycle defects, and cell death. Thus an understanding of factors regulating histone mRNA translation will contribute to our understanding of cancer.