RNA-Binding Proteins that Modulate Proliferative and Stress-Response Genes

调节增殖和应激反应基因的 RNA 结合蛋白

• 批准号: 7732228
• 负责人: MYRIAM none GOROSPE
• 金额: $ 59.21万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732228
• 关键词: 14-3-3 Proteins; Affect; Binding; Biological Assay; Biotin; CDC2 Protein Kinase; Cell Nucleus; Cells; Cultured Cells; EMSA; Funding; Genes; Half-Life; Heat-Shock Response; Heterogeneous Nuclear RNA; Immunoprecipitation; In Vitro; Measures; Messenger RNA; Metabolism; Polyribosomes; Post-Translational Protein Processing; Process; Protein Overexpression; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Rate; Regulation; Relative (related person); Reporter; Reporting; Ribonucleoproteins; Surface Plasmon Resonance; Translating; Translations; Ubiquitination; Work; biological adaptation to stress; insight; interest; mRNA Precursor; mRNA Stability; protein function

RBPs are pivotal regulators of mRNA metabolism, influencing all aspects of post-transcriptional gene control: pre-mRNA splicing, mRNA transport, mRNA stability, and translation. We study if a given RBP associates with a given mRNA by a variety of in vitro binding assays (biotin pulldown, RNA EMSA, surface plasmon resonance/Biacore, etc) and assays to measure binding of endogenous molecules (ribonucleoprotein immunoprecipitation). To investigate RBP function in mammalian cultured cells, we employ approaches such as RBP silencing, RBP overexpression, and the identification of RBP-associated mRNAs using microarrays. We investigate whether RBPs affect the stability of target mRNAs by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs. During this funding period, we have completed two main projects. In one project, we systematically analyzed the interactions of six major RBPs (HuR, AUF1, NF90, KSRP, TIAR, and TIA-1) with the mRNAs that encode these RBPs. We reported that each RBP associates with its own mRNA and uncovered an extensive network of cross-regulation among these RBPs and the mRNAs that encode them. In the other main project, we reported that HuR is phosphorylated by the kinase Cdk1 (Cdc2); Cdk1 phosphorylates HuR at S202 (a residue located within a region important for controlling the subcellular levels of HuR) and together with 14-3-3 proteins, it helped to retain HuR in the nucleus. Ongoing work is examining the subcellular localization of HuR by post-translational modification at S242 (also located within the region that modulates HuR subcellular distribution), and the influence of ubiquitination upon the overall HuR levels in cells responding to heat shock. Other studies are seeking to identify the subsets of mRNAs that are regulated by RBPs NF90 and AUF1.

RBP是mRNA代谢的关键调节剂，影响了转录后基因控制的各个方面：MRNA剪接，mRNA转运，mRNA稳定性和翻译。 我们研究给定的RBP是否通过各种体外结合测定（Biotin pulldown，RNA EMSA，表面等离子体共振/BIACORE等）与给定的mRNA相关联，并测量内源性分子的结合（核糖核蛋白蛋白免疫蛋白免疫蛋白）。 为了研究哺乳动物培养细胞中的RBP功能，我们采用了RBP沉默，RBP过表达等方法，并使用微阵列鉴定了与RBP相关的mRNA。 我们研究了RBP是否通过测量感兴趣的mRNA的稳态水平和半衰期作为RBP丰度的函数来影响目标mRNA的稳定性。 我们研究了RBP是否通过研究mRNA与翻译多元素的相对关联并量化编码蛋白的新生翻译速率来影响目标mRNA的翻译。 我们还采用记者构造来获得对RBPS调制过程的进一步见解。 在此资助期间，我们完成了两个主要项目。 在一个项目中，我们系统地分析了六个主要RBP（HUR，AUF1，NF90，KSRP，TIAR和TIA-1）与编码这些RBP的mRNA的相互作用。 我们报告说，每个RBP都与自己的mRNA相关联，并发现了这些RBP和编码它们的mRNA之间的广泛交叉调节网络。 在另一个主要项目中，我们报道了HUR被激酶CDK1（CDC2）磷酸化。 CDK1磷酸化S202（位于控制HUR的亚细胞水​​平重要的区域内的残基），并与14-3-3蛋白一起，有助于保留核中的HUR。 正在进行的工作正在检查S242的翻译后修饰（也位于调节HUR亚细胞分布的区域内），并检查了HUR的亚细胞定位，以及泛素化对热休克响应的细胞总体HUR水平的影响。 其他研究正在寻求识别由RBPS NF90和AUF1调节的mRNA子集。

项目成果

Identification of a signature motif in target mRNAs of RNA-binding protein AUF1.

• DOI: 10.1093/nar/gkn929
• 发表时间: 2009-01
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Mazan-Mamczarz K;Kuwano Y;Zhan M;White EJ;Martindale JL;Lal A;Gorospe M
• 通讯作者: Gorospe M