Intracellular Traffic of Small Nuclear RNA to the Nucleolus

核仁是一个高度动态的亚细胞室，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，这是一个鲜为人知的功能。该项目将确定U6 snRNA在装配到剪接体之前通过核仁的途径和机制。因此，该项目的结果应该为核仁功能的这方面提供新的和重要的见解。将携带各种突变的荧光素标记的U6 snRNA转录本注射到爪蟾卵母细胞中，鉴定出U6 snRNA核仁定位或核仁输出所需的序列，随后在显微镜下分析核仁制备。一个包含U6 snRNA中已识别的核仁定位元件（NoLEs）与不相关序列融合的迷你构建体将揭示被识别为NoLEs的序列是否不仅是核仁定位所必需的，而且是充分的。将研究U6核仁输出序列与snoRNA Box C/D末端干基序的融合构建。引物延伸试验将用于阐明U6的核仁定位是否需要其修饰（2'- o -甲基化和假尿嘧啶化）。将检测内源性U6的修饰，以及将合成野生型U6和合成突变型U6（不能定位到核仁）注射到缺乏内源性U6 snRNA的卵母细胞中。一个包含被引导snoRNA mgU6-77甲基化的U6区域与40 nt不相关序列融合的迷你构建体将在引导snoRNA耗尽后进行核仁定位测试，以研究引导snoRNAs是否在U6 snRNA核仁定位中发挥作用。将荧光素标记的U4或U5 snrna的合成副本注射到非洲爪蟾卵母细胞中，以检查它们是否在核仁中短暂定位。如果是这样，反义寡核苷酸介导的U6 snRNA缺失将揭示U4和U5 snRNA的核核定位是否需要U6的存在，或者相反，如果U4或U5 snRNA缺失，U6核核定位是否需要U4或U5 snRNA。同一组实验也将在注射后更长的时间点进行，以了解U6 snRNA的核核输出是否需要U4和/或U5 snRNP，并分析输出是否仅在双snRNP和/或三snRNP形成后才发生。La蛋白可能通过结合其NoLEs介导U6 snRNA的核仁定位。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会转化为3’磷酸以阻止与La结合，从而可以分析U6 snRNA核核定位是否需要La结合。如果是这样，将确定La内将该蛋白靶向到核仁的特定区域，以及La磷酸化状态是否对核仁的瞬时定位很重要。

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.