The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis

Abstract

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.

ACKNOWLEDGMENTS

We thank John Gurdon and Kazutaka Murata for providing demembranated Xenopus laevis sperm chromatin and Tony Mills for initial advice on Xenopus egg extract preparation and on the Xenopus DNA replication system. We also thank Alexander Langley, John Gurdon, Olivier Hyrien, and Tony Mills for critical reading and discussions.

This work was supported by Cancer Research UK (project grant C1471/A8448), the UK Medical Research Council (program number U117597140) and the Wellcome Trust. The University of Cambridge Department of Zoology confocal suite was financed by the Wellcome Trust and the Newton Trust.