Kinase-mediated assembly of the mRNA entry channel in 40S ribosomes

 描述(申请人提供)：快速分裂的细胞必须每分钟产生2000个新的核糖体，并确保它们具有完全的功能。MRNA进入通道形成在核糖体小亚基的喙结构上，喙上的三个蛋白质中有两个与DBA有关，DBA是一种遗传性人类疾病，由核糖体蛋白(r-蛋白)单倍体不足引起。正如r-蛋白丢失所预期的那样，患者在快速分裂的组织中细胞数量较少，但也容易患癌症，这表明这些患者无法产生所需的核糖体数量和质量。我们最近的数据表明，在mRNA进入通道的r-蛋白在成熟过程中后期组装，这种组装受组装因子Ltv1的释放调节，这需要Hrr25激酶的磷酸化。该项目的总体目标是使用结构和生化方法剖析mRNA进入通道的组装。在目标1中，我们将剖析Ltv1解离如何影响前40s亚单位的结构，以及mRNA结合通道。我们还将测试Ltv1的释放如何与核糖体蛋白Asc1的招募有关，Asc1是翻译包含IRES的病毒消息所必需的。在目标2中，我们将使用结构和酵母生化方法来定义如何通过参与DBA的蛋白质网络将Rps10在喙上的掺入与18S rRNA在平台上的加工联系起来。这些研究将提供对细胞内非常快速的组装事件的洞察，并增加我们的 了解核糖体成熟缺陷导致肿瘤发生的机制。

 DESCRIPTION (provided by applicant): Rapidly dividing cells must produce 2,000 new ribosomes every minute and ensure that they are fully functional. The mRNA entry channel is formed on the beak structure of the small ribosomal subunit, and two out of the three proteins at the beak are associated with DBA, an inherited human disease, caused by haploinsufficiency of ribosomal proteins (r-proteins). As expected from loss of r-proteins, patients have low cell numbers in rapidly dividing tissues, but are also predisposed to cancer, suggesting that these patients fail in producing both the numbers and quality of ribosomes needed. Our recent data indicates that r- proteins at the mRNA entry channel are assembled late during maturation, and that assembly is regulated by release of the assembly factor Ltv1, which requires phosphorylation by the Hrr25 kinase. The overall goal of this project is to dissect the assembly of the mRNA entry channel using structural and biochemical methods. In Aim 1 we will dissect how Ltv1 dissociation affects the structure of pre-40S subunits, and the mRNA binding channel. We will also test how release of Ltv1 is linked to recruitment of the ribosomal protein Asc1, which is required for translation of IRES-containing viral messages. In Aim 2 we will use structural and yeast biochemical methods to define how incorporation of Rps10 at the beak is linked to 18S rRNA processing at the platform, via a network of proteins involved in DBA. These studies will provide insight into assembly events that are very rapid within cells and increase our understanding of the mechanisms by which defects in ribosome maturation can lead to tumorigenesis.