Amino Acid Regulation of Alternative Splicing

选择性剪接的氨基酸调控

• 批准号: 8520000
• 负责人: MICHAEL S. KILBERG
• 金额: $ 30.51万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8520000
• 关键词: Address; Alternative Splicing; Amino Acids; Anti-Inflammatory Agents; Anti-inflammatory; Area; Arginine; Aspartate-Ammonia Ligase; Biological Assay; CCAAT-Enhancer-Binding Proteins; Cell Culture Techniques; Cell physiology; Cells; Cellular Stress; Complementary DNA; Cultured Cells; Data; Diabetes Mellitus; Diet; Dietary Proteins; Disease; Dominant-Negative Mutation; EMSA; Embryo; Essential Amino Acids; Exhibits; Exons; Feedback; Fetal Development; Fibroblasts; Gene Targeting; Genes; Genetic Transcription; Health; Histidinol; Human; Hybrids; Individual; Inflammation; Inflammatory; Informatin; Investigation; Kinetics; Knock-out; Knowledge; Length; Leucine Zippers; Longevity; MAP Kinase Gene; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Messenger RNA; Microarray Analysis; Monitor; Mus; Nutrient; Pathway interactions; Phosphorylation; Play; Protein Array; Protein Isoforms; Protein Microarray Assay; Protein Microchips; Protein-Restricted Diet; Proteins; RNA; RNA Splicing; Recruitment Activity; Regulation; Reporter Genes; Reporting; Research; Response Elements; Role; Serine; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Testing; Tetanus Helper Peptide; Transgenic Mice; Tumor Promoters; Yeasts; acid stress; acronyms; activating transcription factor; chromatin immunoprecipitation; deprivation; feeding; gene repression; hepatoma cell; human ARMET protein; human FRAP1 protein; hypertensive heart disease; in vivo; insight; knock-down; mRNA Precursor; novel; nutrition; programs; protein intake; protein protein interaction; research study; response; transcription factor; tumor

DESCRIPTION (provided by applicant): Limiting dietary protein intake results in amino acid deficiency within cells and activates several signal transduction pathways collectively called the amino acid response (AAR). A number of genes have been identified that are transcriptionally-activated by the AAR, including the bZIP transcription factor ATF3, for which cellular stress induces multiple isoforms by pre-mRNA alternative splicing. Two of these isoforms, full- length ATF3 (ATF3-FL) and a form with a truncated leucine zipper, ATF3?Zip3, are induced in expression by low protein diet in vivo or by amino acid deprivation of cultured cells. These two isoforms exhibit opposing action on the AAR target gene encoding asparagine synthetase (ASNS); exogenous ATF3-FL expression causes transcriptional repression of the amino acid-dependent induction of ASNS, whereas ATF3?Zip3 further enhances the induction. How the cellular amino acid content signals to and controls pre-mRNA alternative splicing has not been investigated. In fact, the study of the regulation of alternative splicing by macro-nutrients represents an entirely new area of investigation in the splicing field. The hypothesis is that ATF3 isoforms have opposing actions within the cellular response to protein/amino acid stress and that the individual isoforms interact with activity-modifying proteins and/or transcriptional co- regulators that support these opposing activities. To address this global hypothesis, three sub- hypotheses will be tested. Hypothesis I: There are differences in the synthesis and functional activities of specific ATF3 isoforms induced by dietary low protein in mice and amino acid deprivation of cultured cells. The proposed research will investigate the kinetics of synthesis for ATF3-FL and ATF3?Zip3 and the functional consequences of each isoform will be addressed by RNA and protein microarray analysis in transgenic mice expressing either ATF3-FL or ATF3?Zip3 individually. Hypothesis II: Amino acid-dependent signaling pathways regulate the alternative splicing of ATF3 during the AAR. These studies will determine the signaling pathway responsible for sensing and transducing the amino acid deficiency signal to the proteins that regulate exon choice during alternative splicing. Hypothesis III: Protein-protein interactions of individual ATF3 isoforms modulate their action on AAR target genes. ATF3-interacting proteins will be identified and their role in the AAR determined. Collectively, the proposed studies will provide novel information and address significant gaps in our knowledge of ATF3 alternative splicing and ATF3 isoform function. The insight gained from these studies will impact the fields of: 1) macro-nutrient control of pre- mRNA alternative splicing; 2) amino acid-dependent control of transcription; and 3) ATF3 function in nutrition and disease.

描述（由申请人提供）：限制膳食蛋白质摄入量会导致细胞内氨基酸缺乏，并激活多种信号转导途径，统称为氨基酸反应（AAR）。已鉴定出许多由 AAR 转录激活的基因，包括 bZIP 转录因子 ATF3，细胞应激通过前 mRNA 选择性剪接诱导多种亚型。这些同种型中的两种，即全长ATF3 (ATF3-FL)和具有截短的亮氨酸拉链的形式，ATF3?Zip3，通过体内低蛋白饮食或通过培养细胞的氨基酸剥夺来诱导表达。这两种亚型对编码天冬酰胺合成酶 (ASNS) 的 AAR 靶基因表现出相反的作用；外源ATF3-FL表达导致ASNS氨基酸依赖性诱导的转录抑制，而ATF3?Zip3进一步增强诱导。细胞氨基酸含量如何向 mRNA 前体发出选择性剪接信号并对其进行控制尚未得到研究。事实上，常量营养素对选择性剪接的调节研究代表了剪接领域的一个全新的研究领域。假设ATF3同种型在细胞对蛋白质/氨基酸应激的反应中具有相反的作用，并且各个同种型与支持这些相反活性的活性修饰蛋白和/或转录辅助调节剂相互作用。为了解决这个全局假设，将测试三个子假设。假设一：小鼠低蛋白饮食和培养细胞氨基酸剥夺诱导的特定ATF3亚型的合成和功能活性存在差异。拟议的研究将调查 ATF3-FL 和 ATF3?Zip3 的合成动力学，并且将通过在单独表达 ATF3-FL 或 ATF3?Zip3 的转基因小鼠中进行 RNA 和蛋白质微阵列分析来解决每种亚型的功能后果。假设 II：氨基酸依赖性信号通路在 AAR 过程中调节 ATF3 的选择性剪接。这些研究将确定负责感知氨基酸缺乏信号并将其转导至在选择性剪接过程中调节外显子选择的蛋白质的信号传导途径。假设 III：各个 ATF3 同工型的蛋白质-蛋白质相互作用调节其对 AAR 靶基因的作用。 ATF3 相互作用蛋白将被鉴定并确定它们在 AAR 中的作用。总的来说，拟议的研究将提供新颖的信息，并解决我们在 ATF3 选择性剪接和 ATF3 同工型功能方面的知识中的重大差距。从这些研究中获得的见解将影响以下领域：1）前体 mRNA 选择性剪接的宏观营养控制； 2）氨基酸依赖性转录控制； 3) ATF3 在营养和疾病中的功能。