BIOCHEMICAL AND TRANSLATION CONTROL OF CYTOKINE MRNA DEC

这一建议针对的是一个新兴但知之甚少的遗传控制领域，即哺乳动物细胞中短暂的mRNAs的细胞质选择性降解，它受mRNA3‘非编码区(ARE-mRNA)中一个富含AU的元件(ARE)调控。ARE促进mRNA快速衰退的机制，翻译和ARE-mRNA快速衰退之间的相互作用，以及促进ARE-mRNAs选择性快速降解的细胞蛋白质是这一应用的主题。目的1将表征翻译在促进含有GM-CSF ARE的mRNAs快速降解中所起的作用。ARE-mRNAs的翻译已被证明可以加速或激活它们的快速衰退。有人建议进行研究，以探讨翻译激活或促进ARE-mRNAs快速衰退的机制。一组研究将调查ARE-mRNA的翻译如何促进其自身的快速衰退。其他研究将解决是否必须持续合成短暂的反式作用细胞蛋白来促进ARE-mRNA周转。目的2研究ARE-mRNAs快速降解的分子机制。基本上，对控制哺乳动物细胞中ARE mRNAs快速降解的基本分子机制一无所知。被称为AUF1的特异性ARE结合蛋白家族促进ARE-mRNAs的快速衰退。AUF1与mRNA结合，在体内与多种蛋白质形成复合体，包括翻译起始因子eIF4G、聚(A)结合蛋白(PABP)和热休克蛋白HSP70和hsc70。实验旨在了解AUF1与ARE-mRNAs的结合如何促进ARE-mRNAs的快速衰退，以及eIF4G、PAPB和HSP-hsc70在ARE-mRNAs衰退中的作用。AIM 3将鉴定未知的AUF1结合蛋白，并研究它们在调节ARE mRNAs快速衰退中的作用。虽然研究已经确定了几个AUF1结合蛋白，但还在体内检测到了其他结合蛋白，但尚未确定，建议进行实验，使用酵母双杂交分析从基因上鉴定剩余的AUF1结合蛋白，并从生化角度研究这些蛋白在促进或抑制ARE-mRNAs衰退方面的可能作用。

This proposal is directed to an emerging but poorly understand area of genetic control, the selective cytoplasmic degradation of short-lived mRNAs in mammalian cells, which is regulated by an AU-rich element (ARE) found in the 3' non-coding region (3'NCR) of the mRNA (ARE- mRNA). The mechanisms by which the ARE promotes rapid mRNA decay, the interplay between translation and rapid ARE-mRNA decay, and the cellular proteins that promote selective rapid degradation of ARE- mRNAs are the subject of this application. Aim 1 will characterize the role translation plays in promoting rapid degradation of mRNAs containing the GM-CSF ARE. Translation of ARE-mRNAs has been shown to accelerate or activate their rapid decay. Studies are proposed to investigate the mechanism by which translation activates or promotes rapid decay of ARE-mRNAs. One group of studies will investigate how translation of the ARE-mRNA promotes its own rapid decay. Other studies will address whether short-lived trans-acting cellular proteins must be continuously synthesized to promote ARE- mRNA turnover. Aim 2 will investigate the molecular mechanism by which ARE-mRNAs are targeted for rapid degradation. Essentially nothing is known about the fundamental molecular mechanisms that control rapid degradation of ARE mRNAs in mammalian cells. The specific ARE binding protein family known as AUF1 promotes the rapid decay of ARE-mRNAs. AUF1 bound to mRNA forms a complex in vivo with a number of proteins, including translation initiation factor eIF4G, poly(A) binding protein (PABP) and heat shock proteins hsp70 and hsc70. Experiments are proposed to understand how binding of AUF1 to the ARE facilities the rapid decay of ARE-mRNAs, and the roles of eIF4G, PAPB and hsp- hsc70 in ARE-mRNA decay. Aim 3 will identify unknown AUF1 binding proteins and examine their roles in regulating rapid decay of ARE mRNAs. Although studies have identified several AUF1 binding proteins, still other binding prote3ins have been detected in vivo but not identified Experiments are proposed to genetically identify remaining AUF1 binding proteins using a yeast 2- hybrid analysis, and to biochemically investigate possible roles of these proteins in promoting or inhibiting decay of ARE-mRNAs.