BIOGENESIS AND TRAFFICKING OF U3 SMALL NUCLEOLAR RNPS

描述(改编自应用程序)：PI建议研究所有 转录后步骤参与U3小核仁的生物发生 核糖核蛋白颗粒(U3 SnoRNP)。这些步骤包括绑定 U3 snoRNA前体和成熟形式的蛋白质；超甲基化 5‘帽；RNA、RNP中间体和成熟体的核保留 SnoRNP；最后，将U3 snoRNP从核质重新定位到 核仁。选择的实验系统将是非洲爪哇的卵母细胞 出于所有常见的原因。特别是，显微注射的U3 snoRNA 重组成U3 snoRNP，并对其亚细胞定位进行了研究 由此产生的RNPs(细胞质、核质、核仁)都很容易确定 通过生化分级和传统的光学显微镜。这个 将使用非洲爪哇测试系统来确定蛋白质组成 重组的U3 snoRNPs，以及不同的U3 snoRNA序列在 蛋白质结合、snoRNP重组和核仁定位。U3 将通过分析分析鉴定snoRNP蛋白(交联物， 亲和力选择)，但由两个杂交筛选克隆(对于那些可能 与纤毛素交互)和三个混合屏幕(适用于那些可能 直接与U3 snoRNA相互作用)。

DESCRIPTION (adapted from application): The PI proposes to study all posttranscriptional steps involved in biogenesis of the U3 small nucleolar ribonucleoprotein particle (U3 snoRNP). These steps include binding of proteins to the precursor and mature forms of U3 snoRNA; hypermethylation of the 5' cap; nuclear retention of the RNA, RNP intermediates, and the mature snoRNP; and finally, relocalization of the U3 snoRNP from the nucleoplasm to the nucleolus. The experimental system of choice will be the Xenopus oocyte for all the usual reasons. In particular, microinjected U3 snoRNAs are reconstituted into U3 snoRNPs, and the subcellular localization of the resulting RNPs (cytoplasm, nucleoplasm, nucleoli) are easily determined both by biochemical fractionation and by traditional light microscopy. The Xenopus assay system will be used to determine the protein composition of reconstituted U3 snoRNPs, and the role of various U3 snoRNA sequences in protein binding, snoRNP reconstitution, and nucleolar localization. U3 snoRNP proteins will be identified by analytical assays (crosslinking, affinity selection) but cloned by two hybrid screens (for those that might interact with fibrillarin) and three hybrid screens (for those that might interact directly with U3 snoRNA).