Examination of Ornithine Decarboxylase Antizyme RNA Structure and Function from Various Organisms for the Development of Antibiological Agents

项目总结 几乎所有的生物都有能力合成天然的多胺--腐胺、亚精胺和 精胺--细胞生长和分化所必需的物质。由于多胺具有相互作用的能力 几乎每一种生物分子--DNA、RNA、磷脂、蛋白质和三磷酸腺苷等--都扮演着许多角色 在细胞内发挥作用，以支持细胞生长。已经有充分的证据表明，体内的多胺水平 哺乳动物细胞与细胞生长速度相关，在体内观察到了高浓度的多胺 在生长缓慢或静止的细胞中，检测到快速增殖的细胞和低浓度的细胞。不 令人惊讶的是，多胺的运输和代谢受到复杂反馈的高度调控 机械装置。鸟氨酸脱羧酶(ODC)是多胺生物合成的关键调节酶。ODC 动态平衡影响细胞生长和癌症的发展。已观察到许多ODC过度表达 肿瘤类型，包括前列腺癌、乳腺癌和皮肤癌。ODC和细胞对多胺的摄取是 被鸟氨酸脱羧酶抗酶(OAZ)抑制。利用OAZ mRNA制备抗酶蛋白的研究 需要在高度保守的位置进行翻译移码，以绕过过早终止。哺乳动物 OAZ mRNAs还在+1移码位点具有一个假结(PK)RNA3+，可刺激+1移码。 此外，多胺刺激移帧，从而提供了一种反馈机制，从而使 代谢产物的积累会抑制生物合成。尽管OAZ伪结RNA的作用 多胺依赖移码中的元件(进一步命名为OAZ-PK)已被研究，但尚未 被认为是一种独特的多胺“传感器”。核糖开关是位于非编码区的元件 直接与细胞代谢物结合并调节基因表达的mRNA。许多核糖开关提供一种 同源物生物合成途径中基因产物的反馈调节机制 代谢物。核糖开关广泛存在于细菌中，还有一类进一步存在于真菌和植物中，但 在动物身上还没有发现核糖开关。有人认为OAZ-PK RNA具有核糖开关的功能， 并且这里提供的证据是该非编码RNA是多胺传感器。这种RNA元素具有很高的 在精胺生物合成所需的脊椎动物基因中保守。靶向药物的开发 因此，来自不同生物体的假定的精胺核糖开关可能用于广泛的目的 例如抗癌药物、抗真菌药物或杀虫剂。这项提案将审查结构和 来自各种生物体的OAZ-PK RNA的功能，包括那些与生物医学相关的生物-人类， 病原真菌和病虫害-有以下具体目的：(1)检查其特异性 以及多胺与来自不同生物的OAZ RNA的亲和力，(2)研究三维 OAZ RNAs的结构，以及(3)探索不同生物体抗酶OAZ RNAs在控制赤霉病中的作用 基因表达。

PROJECT SUMMARY Nearly all organisms possess the capability to synthesize the natural polyamines - putrescine, spermidine and spermine - which are essential for cell growth and differentiation. Due to the ability of polyamines to interact with nearly every biomolecule - DNA, RNA, phospholipids, proteins and ATP, to name a few - they play many roles within the cell in order to support cell growth. It has been well documented that polyamine levels in mammalian cells correlate with the rate of cell growth, high polyamine concentrations have been observed in rapidly proliferating cells and low concentrations have been measured in slow-growing or quiescent cells. Not surprisingly, the transport and metabolism of polyamines are highly regulated by complex feedback mechanisms. Ornithine decarboxylase (ODC) is the key regulatory enzyme in polyamine biosynthesis. ODC homeostasis affects cell growth and cancer development. ODC over-expression has been observed in many tumor types, including prostate, breast, and skin cancers. Both ODC and cellular uptake of polyamines is inhibited by Ornithine Decarboxylase Antizyme (OAZ). The making of Antizyme protein from OAZ mRNA requires translational frameshifting at a highly conserved site to bypass premature termination. Mammalian OAZ mRNAs further possess a pseudoknot (PK) RNA 3¢ to the frameshift site that stimulates +1 frameshifting. Moreover, frameshifting is stimulated by polyamines, thus providing a feedback mechanism whereby the accumulation of metabolic products inhibits biosynthesis. Although the role of the OAZ pseudoknot RNA element (further designated OAZ-PK) in polyamine-dependent frameshifting has been investigated, it has not been examined as a distinct polyamine “sensor”. Riboswitches are elements within noncoding regions of mRNAs that directly bind to cellular metabolites and modulate gene expression. Many riboswitches provide a mechanism of feedback regulation for gene products within the biosynthetic pathway of the cognate metabolite. Riboswitches are widespread among bacteria, and one class further resides in fungi and plants, but no riboswitches have been found in animals. It is proposed that the OAZ-PK RNA functions as a riboswitch, and herein evidence is provided that this noncoding RNA is a polyamine sensor. This RNA element is highly conserved among vertebrate genes required for spermine biosynthesis. Development of drugs that target putative spermine riboswitches from different organisms might therefore be used for wide-ranging purposes such as anticancer agents, antifungal agents, or pesticides. This proposal will examine the structure and function of the OAZ-PK RNA from various organisms, including those of biomedical relevance – human, pathogenic fungi and disease harboring insects - with the following specific aims: (1) examine the specificity and affinity of polyamine binding to OAZ RNAs from various organisms, (2) investigate the three-dimensional structure of OAZ RNAs, and (3) explore the role of Antizyme OAZ RNAs from various organisms in control of gene expression.