MicroRNAs Influencing the Expression of Proliferative and Stress-Response Genes

影响增殖和应激反应基因表达的 MicroRNA

• 批准号: 7732227
• 负责人: MYRIAM none GOROSPE
• 金额: $ 16.33万
• 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/7732227
• 关键词: 3' Untranslated Regions; Affect; Affinity; Aging; Area; Binding; Biology; Cell Aging; Cell Cycle Progression; Cells; Code; Event; Funding; Gene Expression; Genes; Half-Life; Investigation; Laboratories; Mammalian Cell; Measures; Messenger RNA; Methodology; MicroRNAs; Polyribosomes; Process; Protein Overexpression; Proteins; Rate; Relative (related person); Reporter; Reporting; Research; Stress; System; Transcript; Translating; Translations; Tumor Suppressor Proteins; Work; biological adaptation to stress; c-myc Genes; insight; interest; mRNA Stability; protein expression; response; senescence

The area of microRNA biology is relatively new in our laboratory. However, we are interested in expanding the scope of our research to include microRNAs, since they are major regulators of gene expression but their influence during the stress and proliferative responses is poorly understood. We are particularly well poised to investigate microRNAs because their effects on mRNA stability and translation are similar in many respects to those of RBPs; therefore, we already have the cell systems, methodologies, and expertise in place to study them. To investigate microRNA levels and function in mammalian cells, we employ approaches such as microRNA overexpression (by transfecting microRNA precursors), microRNA reduction (by transfecting antisense microRNAs), and the identification of microRNA-associated mRNAs using biotinylated microRNAs to affinity-purify bound transcripts. We investigate whether microRNAs affect the stability of target mRNAs by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of microRNA abundance. We investigate whether microRNAs 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 these processes. During this funding period, we have reported that miR-24 reduces the translation of p16 by associating at least with two segments of the p16 mRNA, one in its coding region, one in the 3-untranslated region, a tumor suppressor that blocks cell cycle progression by inhibiting cdk4/6. In the course of this investigation, we also identified other microRNAs whose levels were upregulated or downregulated during replicative senescence. Ongoing work is analyzing the mRNA targets of various senescence-associated microRNAs. Among the targets under study is MKK4, whose translation appears to be modulated jointly by several microRNAs. In addition, we are pursuing the analysis of microRNAs that potently modulates the translation of the RBP HuR. Other studies are focused on the influence of RBPs upon microRNA action and vice versa, using the c-Myc mRNA as the target transcript.

在我们的实验室中，microRNA生物学区域相对较新。 但是，我们有兴趣扩大我们的研究范围以包括microRNA，因为它们是基因表达的主要调节剂，但是它们在压力和增殖反应中的影响很少。 我们特别准备研究microRNA，因为它们对mRNA稳定性和翻译的影响在许多方面与RBP的影响相似。 因此，我们已经有了细胞系统，方法和专业知识来研究它们。 为了研究哺乳动物细胞中的microRNA水平和功能，我们采用了诸如microRNA过表达（通过转染microRNA前体），减少microRNA的减少（通过转染反义microRNAS）以及使用生物素基化的microRNAS与亲密纯化的界限进行了递质的MRNAS鉴定。 我们研究了MicroRNA是否通过测量感兴趣的mRNA的稳态水平和半衰期来影响目标mRNA的稳定性，这是microRNA丰度的函数。 我们研究了microRNA是否通过研究mRNA与翻译多粒子体的相对关联以及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这些过程的更多见解。 在这一资金期间，我们报道了miR-24通过至少将p16 mRNA的两个段关联，一个在其编码区域中，一个在3-非翻译区域中降低了p16的翻译，这是一种抑制肿瘤，该区域通过抑制CDK4/6来阻断细胞周期的进展。 在这项调查过程中，我们还确定了在复制衰老期间上调或下调的其他microRNA。 正在进行的工作正在分析各种衰老相关的microRNA的mRNA靶标。 研究的目标是MKK4，其翻译似乎是由几种microRNA共同调节的。 此外，我们正在对MicroRNA进行分析，该分析有效调节RBP HUR的翻译。 其他研究的重点是RBP对MicroRNA作用的影响，反之亦然，使用C-Myc mRNA作为目标转录本。