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 12(th) 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 modified antisense oligonucleotide targeted to its open reading frame. 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.
Slevin
Michael K
MK
Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa, USA.
Lyons-Levy
Gillian
G
Weeks
Daniel L
DL
Hartley
Rebecca S
RS
D000818:Animals; D001483:Base Sequence; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics* / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014982:Xenopus laevis / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*
D019925:Cyclin A; D019927:Cyclin E; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger