Michael K. Slevin

The F-box protein Cdc4/Fbxw7 is a novel regulator of neural crest development in Xenopus laevis.

BackgroundThe neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this.ResultsHere we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4α and xCdc4β. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail.ConclusionsWe demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation.

ElrA binding to the 3′UTR of cyclin E1 mRNA requires polyadenylation elements

The early cell divisions of Xenopus laevis and other metazoan embryos occur in the presence of constitutively high levels of the cell cycle regulator cyclin E1. Upon completion of the 12th cell division, a time at which many maternal proteins are downregulated by deadenylation and destabilization of their encoding mRNAs, maternal cyclin E1 protein is downregulated while its mRNA is polyadenylated and stable. We report here that stable polyadenylation of cyclin E1 mRNA requires three cis-acting elements in the 3′ untranslated region; the nuclear polyadenylation sequence, a contiguous cytoplasmic polyadenylation element and an upstream AU-rich element. ElrA, the Xenopus homolog of HuR and a member of the ELAV gene family binds the cyclin E1 3′UTR with high affinity. Deletion of these elements dramatically reduces the affinity of ElrA for the cyclin E1 3′UTR, abolishes polyadenylation and destabilizes the mRNA. Together, these findings provide compelling evidence that ElrA functions in polyadenylation and stabilization of cyclin E1 mRNA via binding these elements.

Antisense knockdown of cyclin E does not affect the midblastula transition in Xenopus laevis embryos.

In Xenopus laevis embryos cyclin E protein remains constitutively high throughout the first 12 cell cycles following fertilization until the onset of the midblastula transition (MBT) (after the 12th cell cycle) when it undergoes a dramatic reduction. The disappearance of cyclin E at the MBT occurs independently of active cell cycle progression, zygotic transcription, protein synthesis and the nuclear to cytoplasmic ratio. This has suggested that cyclin E is part of an autonomous maternal timer that regulates the onset of the MBT. To determine how constitutively high levels of cyclin E are maintained prior to the MBT and to investigate if the reduction in cyclin E protein affects the timing of the MBT, we have knocked down endogenous cyclin E mRNA using an N,N-diethyl-ethylene-diamine (DEED) modified antisense oligonucleotide (ODN) targeted to its open reading frame (ORF). We report that maintenance of high levels of cyclin E protein before the MBT is due to a balance between ongoing translation and proteolytic degradation. In support of our antisense experiments, polysome analysis demonstrates that cyclin E mRNA is associated with the translated fraction prior to the MBT. Furthermore, knockdown of cyclin E was not associated with defects in the timing of developmental events. Our data suggests that cyclin E is not required for the later cell cycles of embryonic development and that the pathway effecting downregulation of cyclin E rather then cyclin E degradation itself may be part of a maternal timer that affects the onset of the MBT. 2