Functions of 5' NCRs of picornavirus and cellular mRNAs

小核糖核酸病毒和其他正链RNA病毒利用5‘中的序列 它们基因组RNA的非编码区(5个NCR)与宿主细胞和病毒蛋白结合以携带 在其细胞内复制周期中具有重要的功能。脊髓灰质炎病毒，柯萨奇病毒， 和人类鼻病毒是微小核糖体病毒科的成员，它们共享一个共同的次级RNA 在翻译起始过程中，核糖体进入内部所需的5个NCR中的结构。这些 结构(即IRES元件)与几种宿主RNA结合蛋白结合，包括聚(RC) 结合蛋白2(PCBP2)。脊髓灰质炎病毒翻译启动需要结合PCBP2，a 这一过程还需要核质穿梭蛋白SRp20的功能。这个 这项建议的第一个目标是确定组装的翻译起始复合体的成分 与PCBP2结合的脊髓灰质炎病毒IRES序列。基因和生化实验将测试 SRp20作为PCBP2与脊髓灰质炎病毒结合的分子桥梁的假说 IRES和细胞翻译启动装置的特定元件。除了它在以下方面的作用 脊髓灰质炎病毒翻译，细胞蛋白PCBP2是负链病毒RNA合成所必需的。 在脊髓灰质炎病毒感染HeLa细胞期间，PCBP2被病毒蛋白酶切割产生 截短的蛋白质，不能在翻译中发挥作用，但在RNA复制中保留其作用。 该提案的第二个目标将利用细胞培养和体外翻译/RNA复制 确定这种切割是否负责清除脊髓灰质炎病毒基因组RNA的方法 在病毒RNA合成开始之前翻译核糖体，提供了一个机制开关 用于利用正链RNA模板的这两个相互竞争的功能。来自这些的结果 拟议的研究将为翻译之间的相互作用提供新的机械论见解 功能与微小核糖核酸复制。在这个项目的新方向上，IRES元素 已在两个细胞信使核糖核酸分子的长5？NCR中被鉴定，其中一个编码 电压门控钾通道(Kv1.4)和编码转录因子(Lef-1)的钾通道。 第三个目标中提出的实验将检查形成的RNP复合体的性质 用这两个细胞mRNA5个NCR来定义IRES功能的决定因素 与小核糖核酸不同的是，在细胞核中合成的mRNAs的翻译 在它们与40S核糖体亚基结合之前，必须运输到细胞质。 后一项研究的结果将提供重要的并列比较 微小核糖核酸病毒IRES的功能和对真核细胞如何启动翻译的新见解 具有长的、高度结构的5？NCRs的mRNAs。

Picornaviruses as well as other positive strand RNA viruses utilize sequences in the 5¿ noncoding regions (5¿ NCRs) of their genomic RNAs to bind host cell and viral proteins to carry out important functions during their intracellular replication cycles. Poliovirus, coxsackievirus, and human rhinovirus are members of the Picornaviridae that share a common RNA secondary structure in their 5¿ NCRs required for internal ribosome entry during translation initiation. These structures (i.e., IRES elements) bind several host RNA binding proteins, including poly(rC) binding protein 2 (PCBP2). Binding of PCBP2 is required for poliovirus translation initiation, a process that also requires the function of a nucleo-cytoplasmic shuttling protein, SRp20. The first aim of this proposal is to identify components of translation initiation complexes assembled with PCBP2-bound poliovirus IRES sequences. Genetic and biochemical experiments will test the hypothesis that SRp20 acts as a molecular bridge between PCBP2 bound to the poliovirus IRES and specific elements of the cellular translation initiation apparatus. In addition to its role in poliovirus translation, cellular protein PCBP2 is required for negative-strand viral RNA synthesis. During poliovirus infection of HeLa cells, PCBP2 is cleaved by a viral proteinase to generate a truncated protein that is unable to function in translation but retains its role in RNA replication. The second aim of the proposal will utilize cell culture and in vitro translation/RNA replication approaches to determine if this cleavage is responsible for clearing poliovirus genomic RNAs of translating ribosomes prior to the onset of viral RNA synthesis, providing a mechanistic switch for these two competing functions utilizing positive-strand RNA templates. Results from these proposed studies will provide new mechanistic insights into the interplay between translation functions and picornavirus RNA replication. In a new direction for this project, IRES elements have been identified in the long 5¿ NCRs of two cellular mRNA molecules, one that encodes a voltage-gated potassium channel (Kv1.4) and one that encodes a transcription factor (LEF-1). The experiments proposed in the third aim will examine the nature of RNP complexes formed with the 5¿ NCRs of these two cellular mRNAs to define the determinants of IRES functions for translation of mRNAs that, unlike picornavirus RNAs, are synthesized in the nucleus of cells and must be transported to the cytoplasm prior to their association with 40S ribosomal subunits. Results from these latter studies will provide important side-by-side comparisons with picornavirus IRES functions and new insights into how eukaryotic cells initiate translation from mRNAs with long, highly-structured 5¿ NCRs.