Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cellular Biology
Rebecca S. Hartley
The embryonic cell cycle of Xenopus laevis consists of rapid oscillations between S and M phase occurring in the absence of gap phases and their associated regulatory checkpoints. The end of the 12th cell cycle marks the onset of the midblastula transition (MBT) when the cell cycle lengthens establishing gap phases, their associated checkpoint pathways, and the initiation of zygotic transcription.
During cell cycles 2-12, cyclins A and B are translated and expressed once per cell cycle until the MBT when their protein levels decrease due to a newly transcribed zygotic factor that leads to the deadenylation and subsequent loss of their mRNAs. In contrast, cyclin E is expressed at a constitutively high level during cell cycles 2-12. Furthermore, cyclin E levels are terminally lost coincident with initiation of the MBT in the continued presence of its adenylated mRNA. Terminal disappearance of cyclin E appears to be maternally directed and is not affected by zygotic transcription, translation, replication, or the nuclear to cytoplasmic ratio. This has led to the hypothesis that cyclin E is part of an autonomous maternally directed timer that determines the timing of the MBT. To investigate this possibility we have used antisense oligonucleotides to knockdown cyclin E and assess the affects on the timing of the MBT. Premature knockdown of cyclin E did not affect the timing of the MBT indicating it is not part of the maternal timer. Furthermore, prior to the MBT cyclin E protein has an unusually long half life. However, despite an increased stability the constitutively high levels of cyclin E require a low level of translation.
We have also determined that the stable pattern of adenylation observed for cyclin E1 is specified by three cis-acting elements in its' 3' UTR. Deletion of the NPS, eCPE/ARE3, and ARE2 abolished adenylation. Additionally, a putative stem loop in ARE2 is targeted by ElrA the Xenopus homolog of HuR and a member of the ELAV gene family. Loss of adenylation required disruption of ElrA binding. These findings demonstrate ElrA functions in the correct adenylation of cyclin E1 mRNA.
Copyright 2006 Michael Keith Slevin