Intracellular Traffic of Small Nuclear RNA to the Nucleolus

核仁是一个高度动态的亚细胞器区室，其不同的作用现在才刚刚开始被理解。 其中一个功能涉及剪接体的组装，这是一个知之甚少的功能。 该项目将确定U6 snRNA在组装成剪接体之前穿过核仁的途径和机制。 因此，该项目的结果应提供新的和重要的见解这方面的核仁功能。 通过将携带各种突变的U6 snRNA的荧光素标记转录物注射到非洲爪蟾卵母细胞中，随后通过显微镜分析核仁制备物，鉴定U6 snRNA的核仁定位或核仁输出所需的序列。含有与不相关序列融合的U6 snRNA的经鉴定的核仁定位元件（NoLE）的微型构建体将揭示鉴定为NoLE的序列是否不仅是核仁定位所需的，而且是足够的。将研究U6核仁输出序列与snoRNA Box C/D末端茎基序的融合构建体，并采用引物延伸分析来阐明U6的核仁定位是否需要其修饰（2'-O-甲基化和假尿苷化）。将检查内源性U6、合成野生型U6和合成突变体U6（不能定位于核仁）的修饰，这些修饰已注射到内源性U6 snRNA耗尽的卵母细胞中。将在耗尽引导snoRNA后测试含有U6区域的融合物的微型构建体，所述U6区域被引导snoRNA mgU6 - 77甲基化，具有40 nt的不相关序列，以研究引导snoRNA是否在U6 snRNA核仁定位中起作用。将标记的U4或U5 snRNA注射到爪蟾卵母细胞中以检查它们是否瞬时定位在核仁中。如果是这样的话，反义寡核苷酸介导的U6 snRNA的缺失将揭示U4和U5 snRNA的核仁定位是否需要U6的存在，或者相反地，随着U4或U5 snRNA的缺失，U6核仁定位是否需要U4或U5 snRNA。在注射后的较长时间点也将进行相同的一组实验，以询问U6 snRNA的核仁输出是否需要U4和/或U5 snRNP，并分析输出是否仅在二-snRNP和/或三-snRNP形成后发生。可能通过与其NoLE结合来介导U6 snRNA的核仁定位的候选蛋白是La蛋白。La结合到U6 snRNA的3 '端，在核仁定位所需的区域内。合成的U6 snRNA的3'-OH将被转化为3 '磷酸盐以防止与La结合，从而允许分析La结合是否是U6 snRNA核仁定位所需的。如果是这样，将确定La内将该蛋白质靶向核仁的特定区域，以及La磷酸化的状态对于瞬时定位到核仁是否重要。

PROJECT SUMMARYThe nucleolus is a highly dynamic sub-organellar compartment whose diverse roles are now only beginning to be understood. One such function involves the assembly of spliceosomes, a function poorly understood. This project will determine the pathway and mechanism by which the U6 snRNA travels through the nucleolus prior to its assembly into spliceosomes. The results of this project should thus provide new and important insights into this aspect of nucleolar function. The sequences required for nucleolar localization or nucleolar export of U6 snRNA will be identified by injection into Xenopus oocytes of fluorescein-labeled transcripts of U6 snRNA carrying various mutations with subsequent analysis of nucleolar preparations by microscopy. A mini-construct containing the identified nucleolar localization elements (NoLEs) of U6 snRNA fused to unrelated sequences will reveal if the sequences identified as NoLEs are not only required but also sufficient for nucleolar localization. A fusion construct of U6 nucleolar export sequences and the snoRNA Box C/D terminal stem motif will be studied.Primer extension assays will be employed to elucidate if nucleolar localization of U6 is required for its modification (2'-O-methylations and pseudouridylations). Modification will be examined for endogenous U6, and for synthetic wild type U6 and synthetic mutant U6 (that cannot localize to nucleoli) which have been injected into oocytes depleted of endogenous U6 snRNA. A mini-construct containing a fusion of the region of U6 which becomes methylated by the guide snoRNA mgU6-77 with 40 nt of unrelated sequence will be tested for nucleolar localization after depletion of the guide snoRNA to investigate whether guide snoRNAs play a role in U6 snRNA nucleolar localization.Synthetic copies of fluorescein-labelled U4 or U5 snRNAs will be injected into Xenopus oocytes to examine if they localize transiently in nucleoli. If so, antisense oligonucleotide mediated depletion of U6 snRNA will reveal if U4 and U5 snRNA nucleolar localization require the presence of U6, or, conversely with depletion of U4 or U5 snRNAs, if U6 nucleolar localization requires U4 or U5 snRNAs. The same set of experiments will also be carried out at longer time points after injection to inquire if the nucleolar export of U6 snRNA requires U4 and/or U5 snRNP, and to analyze if export occurs only after the di-snRNP and/or tri-snRNP have formed. A candidate protein that may mediate the nucleolar localization of U6 snRNA by binding to its NoLEs is the La protein. La binds to the 3' end of U6 snRNA, within the region that is required for nucleolar localization. The 3'-OH of synthetic U6 snRNA will be converted to a 3' phosphate to prevent association with La, thus allowing analysis of whether La binding is required for U6 snRNA nucleolar localization. If so, the specific region within La that targets this protein to the nucleolus will be determined, and whether the state of La phosphorylation is important for transient localization to the nucleolus.