EFFECT OF UBF ON NUCLEOLAR STRUCTURE IN XENOPUS EMBRYO DEVELOPMENT

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。UBF是一种高度保守的转录因子，对RNA聚合酶I介导的转录起始至关重要。尽管其明显的特异性，rDNA相关的功能，一些证据表明，UBF可能在其他类别的转录功能。UBF的存在远远超过RNA聚合酶I介导的转录理论上所需的量，位于核仁和核质两者，具有松散的DNA结合特异性，并且可以通过RNA聚合酶II促进某些基因的转录。解决UBF在体内作用的一种方法是下调其蛋白水平。目前还没有UBF基因敲除的报道，因此我们尝试用反义吗啉寡核苷酸敲除非洲爪蟾胚胎中的UBF蛋白水平。非洲爪蟾胚胎是一个理想的模型系统，因为它们的rDNA是沉默的，核仁是不存在的，直到大约第9阶段，从而提供了一个窗口，其中UBF可以下调之前，它是积极的pol I转录。此外，以前的表征无核仁非洲爪蟾胚胎已经失去了超过99%的rDNA重复显示，这些胚胎存活到第40阶段，大概是通过使用母体核糖体。因此，我们预期UBF敲低将导致类似的表型。我们已经成功地和具体地降低了非洲爪蟾胚胎中的UBF蛋白水平，并发现它们在10-16阶段之间意外地冻结发育。通过光学显微镜或电子显微镜测定，降低的UBF水平不影响大多数细胞的核仁结构，并且未观察到rRNA水平的显著降低，表明这些细胞很可能保留了一些RNA聚合酶I活性。虽然整体蛋白质合成没有受到显着影响，但我们有证据表明，参与前rRNA加工的另一种蛋白质（Sof 1）也被下调，可能是次要影响。结合无核仁表型，这些结果表明，UBF可能参与过程以外的聚合酶I转录，这些过程是必不可少的爪蟾胚胎发育。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. UBF is a highly conserved transcription factor that is essential for the initiation of RNA polymerase I-mediated transcription. Despite its apparently specific, rDNA-related function, several lines of evidence suggest that UBF may function in other classes of transcription. UBF is present far in excess of the amount theoretically needed for RNA polymerase I-mediated transcription, is localized to both nucleoli and nucleoplasm, has loose DNA binding specificity, and can promote the transcription of some genes by RNA polymerase II. One way to address the roles of UBF in vivo is to downregulate its protein levels. No gene knockout of UBF has yet been reported so we have attempted to knockdown UBF protein levels in the embryos of Xenopus laevis using antisense morpholino oligonucleotides. Xenopus embryos are an ideal model system since their rDNA is silenced and nucleoli are absent until around stage 9, thus providing a window in which UBF can be downregulated before it is active in pol I transcription. In addition, previous characterization of anucleolar Xenopus embryos which have lost over 99% of their rDNA repeats exhibited that these embryos survive until stage 40, presumably by using maternal ribosomes. We thus expected that UBF knockdown would result is a similar phenotype. We have successfully and specifically reduced UBF protein levels in Xenopus embryos and have found that they unexpectedly freeze in development between stages 10-16. The decreased UBF levels do not affect nucleolar structure in the majority of cells as assayed by light or electron microscopy and no significant decrease in the levels of rRNA was seen, indicating that these cells most likely retain some RNA polymerase I activity. Whereas overall protein synthesis not significantly affected, we have evidence that another protein involved in pre-rRNA processing (Sof1) is also down-regulated, presumably as a secondary effect. In combination with the anucleolar phenotype, these results suggest that UBF may be involved in processes outside of pol I transcription and that these processes are essential in Xenopus embryonic development.