Amino Acid Regulation of Alternative Splicing

描述（由申请人提供）：限制膳食蛋白质摄入会导致细胞内氨基酸缺乏，并激活几种信号转导途径，统称为氨基酸反应（AAR）。许多基因被AAR转录激活，包括bZIP转录因子ATF3，细胞应激通过mrna前选择性剪接诱导多种亚型。其中两种同工异构体，全长ATF3 （ATF3- fl）和具有截断亮氨酸拉链的ATF3？体内低蛋白饮食或培养细胞氨基酸剥夺均可诱导Zip3的表达。这两个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表达可导致氨基酸依赖性ASNS的转录抑制，而ATF3？Zip3进一步增强了诱导性。细胞氨基酸含量如何信号和控制前mrna选择性剪接尚未被研究。事实上，研究大量营养物质对选择性剪接的调控是剪接领域一个全新的研究领域。假设ATF3亚型在细胞对蛋白质/氨基酸应激的反应中具有相反的作用，并且单个亚型与支持这些相反活动的活性修饰蛋白和/或转录共调节因子相互作用。为了解决这个全球性的假设，我们将测试三个子假设。假设一：小鼠饮食低蛋白与培养细胞氨基酸剥夺诱导的ATF3特异性异构体的合成和功能活性存在差异。本研究将研究ATF3- fl和ATF3？在表达ATF3- fl或ATF3- fl的转基因小鼠中，将通过RNA和蛋白质微阵列分析解决Zip3和每个亚型的功能后果。单独zip2。假设二：氨基酸依赖的信号通路在AAR过程中调节ATF3的选择性剪接。这些研究将确定负责感知和转导氨基酸缺乏信号到在选择性剪接过程中调节外显子选择的蛋白质的信号通路。假设三：单个ATF3亚型的蛋白-蛋白相互作用调节其对AAR靶基因的作用。将鉴定atf3相互作用蛋白并确定其在AAR中的作用。总的来说，拟议的研究将提供新的信息，并解决我们在ATF3选择性剪接和ATF3异构体功能方面的重大知识空白。从这些研究中获得的见解将影响以下领域：1)前mRNA选择性剪接的宏观营养控制；2)氨基酸依赖性转录调控；3) ATF3在营养和疾病中的作用。

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.