EFFECT OF UBF ON NUCLEOLAR STRUCTURE IN XENOPUS EMBRYO DEVELOPMENT

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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。尽管它具有明显的特异性，与rdna相关的功能，但一些证据表明，UBF可能在其他类型的转录中起作用。UBF的存在量远远超过RNA聚合酶i介导的转录理论上所需的量，定位于核仁和核质，具有松散的DNA结合特异性，可以促进RNA聚合酶II对某些基因的转录。解决UBF在体内作用的一种方法是下调其蛋白水平。目前还没有关于UBF基因敲除的报道，因此我们尝试用反义morpholino寡核苷酸敲除非洲爪蟾胚胎中UBF蛋白的水平。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，核仁是缺失的，直到大约9期，从而提供了一个窗口，在UBF在pol I转录中活跃之前，它可以下调。此外，先前对失去99%以上rDNA重复序列的无核爪蟾胚胎的表征表明，这些胚胎可能是通过母体核糖体存活到40期。因此，我们预计UBF敲低会导致类似的表型。我们已经成功地专门降低了爪蟾胚胎中的UBF蛋白水平，并发现它们意外地在10-16期之间冻结了发育。通过光镜或电子显微镜检测，UBF水平的降低并不影响大多数细胞的核仁结构，rRNA水平也没有明显下降，这表明这些细胞很可能保留了一些RNA聚合酶I的活性。尽管整体蛋白质合成没有受到显著影响，但我们有证据表明，另一种参与前rrna加工的蛋白质（Sof1）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可能参与了pol I转录之外的过程，这些过程在非洲爪蟾胚胎发育中是必不可少的。

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.