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

参与组蛋白 mRNA 和 PolyA mRNA 子集翻译的新因子

• 批准号: 7579020
• 负责人: RACHEL S. LERNER
• 金额: $ 1.49万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7579020
• 关键词: Affect; Binding; Binding Proteins; Biochemical; Cell Cycle; Cell Death; Cell physiology; Collaborations; Complex; DNA Damage; DNA biosynthesis; Data; Defect; Elements; Elongation Factor; Ensure; Essential Genes; Eukaryotic Initiation Factors; Fibrinogen; Genetic Translation; Goals; Heating; Histones; Immunoprecipitation; Laboratories; Lead; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Microarray Analysis; Molecular; Mutation; Names; Phase; Play; Poly A; Poly(A) Tail; Poly(A)-Binding Proteins; Process; Production; Protein Binding; Proteins; RNA Interference; RNA-Binding Proteins; Reporter; Repression; Ribosomes; Role; Structure; Techniques; Testing; Translation Process; Translational Activation; Translations; Universities; Xenopus oocyte; Yeasts; in vivo; novel; stem; tissue/cell culture; translation assay; translation factor; tumorigenesis; yeast two hybrid system

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.

描述（申请人提供）：组蛋白mRNA水平与细胞周期协调调节，在S期开始时积累并在S期结束时降解。这些后生动物复制依赖性组蛋白 mRNA 是独特的，因为与其他 mRNA 不同，它们不是聚腺苷酸化的，而是以保守的茎环结构结尾。这个独特的 3' 末端与茎环结合蛋白 (SLBP) 结合，在组蛋白 mRNA 加工、翻译和降解中发挥作用。我的研究重点是了解 Marzluff 实验室发现的一种与 SLBP 结合的新型蛋白质（名为 SLIP1）的作用。初步数据表明，SLIP1 参与组蛋白 mRNA 翻译，并可能参与一部分 Poly (A) mRNA 的翻译，并且是一个必需基因。该提案的广泛长期目标是定义 SLIP1 作为翻译因子的作用的分子细节，并确定除组蛋白 mRNA 之外，哪些细胞 mRNA 需要 SLIP1 进行翻译。为了将 SLIP1 描述为翻译因子，我将采用分子和生化技术。使用非洲爪蟾卵母细胞和哺乳动物细胞的体内翻译测定将确定对于 SLIP1 介导的翻译很重要的 mRNA 顺式元件。其他生化方法将用于检查 SLIP1 与一般翻译因子和核糖体的结合。为了鉴定 SLIP1 结合蛋白，我将使用 SLIP1 作为诱饵进行酵母双杂交筛选，并对 SLIP1 进行免疫沉淀，然后进行质谱分析。鉴定出的蛋白质将通过翻译测定进行验证，以确认 SLIP1 和鉴定出的蛋白质之间的功能相互作用。通过 SLIP1-RNP 复合物的免疫沉淀，然后对沉淀的 mRNA 进行微阵列分析，可以鉴定受 SLIP1 翻译调节的细胞 mRNA。微阵列分析将通过与 Michael Whitfield 博士实验室（达特茅斯大学）的合作完成，该实验室拥有微阵列分析方面的专业知识，并且之前曾与 Marzluff 实验室合作过。通过这种方法鉴定的 mRNA 将通过体内翻译测定进行测试，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 DNA 合成过程中组蛋白丰富。导致组蛋白生产缺陷的突变可能导致肿瘤发生、DNA 损伤、细胞周期缺陷和细胞死亡。因此，了解调节组蛋白 mRNA 翻译的因素将有助于我们了解癌症。

项目成果

Role of oligouridylation in normal metabolism and regulated degradation of mammalian histone mRNAs.

寡尿苷化在正常代谢和调节哺乳动物组蛋白 mRNA 降解中的作用。

• 发表时间: 2018
• 作者: Meaux, Stacie A;Holmquist, Christopher E;Marzluff, William F
• 通讯作者: Marzluff, William F

Structural and biochemical studies of SLIP1-SLBP identify DBP5 and eIF3g as SLIP1-binding proteins.

SLIP1-SLBP 的结构和生化研究确定 DBP5 和 eIF3g 为 SLIP1 结合蛋白。

• 发表时间: 2013-09
• 影响因子: 14.9
• 作者: von Moeller H;Lerner R;Ricciardi A;Basquin C;Marzluff WF;Conti E
• 通讯作者: Conti E

Deep sequencing shows multiple oligouridylations are required for 3' to 5' degradation of histone mRNAs on polyribosomes.

• DOI: 10.1016/j.molcel.2014.02.027
• 发表时间: 2014-03-20
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Slevin, Michael K.;Meaux, Stacie;Welch, Joshua D.;Bigler, Rebecca;de Marval, Paula L. Miliani;Su, Wei;Rhoads, Robert E.;Prins, Jan F.;Marzluff, William F.
• 通讯作者: Marzluff, William F.