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.
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