Functions of 5' NCRs of picornavirus and cellular mRNAs

小核糖核酸病毒以及其他正链RNA病毒利用5 '中的序列 它们基因组RNA的非编码区（5 <$NCR）结合宿主细胞和病毒蛋白， 在细胞内复制周期中发挥重要作用。脊髓灰质炎病毒柯萨奇病毒， 和人鼻病毒是小核糖核酸病毒科的成员， 在翻译起始过程中进入内部核糖体所需的5 <$NCR结构。这些 结构（即，IRES元件）结合几种宿主RNA结合蛋白，包括poly（rC） 结合蛋白2（PCBP 2）。PCBP 2的结合是脊髓灰质炎病毒翻译起始所必需的， 该过程还需要核质穿梭蛋白SRp 20的功能。的 该建议的第一个目的是鉴定组装的翻译起始复合物的组分 与PCBP 2结合的脊髓灰质炎病毒IRES序列。基因和生化实验将测试 SRp 20作为PCBP 2与脊髓灰质炎病毒结合的分子桥梁的假设 IRES和细胞翻译起始装置的特定元件。除了在以下方面的作用外， 在脊髓灰质炎病毒翻译过程中，细胞蛋白PCBP 2是负链病毒RNA合成所必需的。 在脊髓灰质炎病毒感染HeLa细胞的过程中，PCBP 2被病毒蛋白酶切割，产生一种蛋白酶。 在翻译中不能起作用但保留其在RNA复制中作用的截短蛋白质。 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，提供了一个机械开关， 利用正链RNA模板来实现这两种竞争功能。从这些 建议的研究将提供新的机制的见解之间的相互作用翻译 功能和小核糖核酸病毒RNA复制。在这个项目的一个新方向，IRES元素 已经在两种细胞mRNA分子的长5 <$NCR中鉴定出，其中一种编码一种 电压门控钾通道（Kv1.4）和一个编码转录因子（LEF-1）。 在第三个目标中提出的实验将检查所形成的RNP复合物的性质 与这两种细胞mRNA的5 <$NCR一起定义IRES功能的决定因素， 与小核糖核酸病毒RNA不同，mRNA在细胞核中合成， 必须在与40 S核糖体亚基结合之前转运到细胞质。 后面这些研究的结果将提供与以下研究的重要并排比较 小核糖核酸病毒IRES的功能和新的见解，真核细胞如何启动翻译从 具有长的、高度结构化的5个NCR的mRNA。

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.