MODIFIED MESSENGER-RNA RESCUE OF MATERNAL CK1 8 MESSENGER-RNA DEPLETION IN XENOPUS OOCYTES/

This work addresses two issues, the use of antisense oligodeoxynucleot ides to deplete specific mRNAs in Xenopus oocytes to analyze their fun ctions during development and the role of cytokeratin filaments in cel ls of the early Xenopus embryo. We have shown previously that depletio n of cytokeratin CK1/8 mRNA causes defects in the early embryo, In thi s study, we show that the oligos, modified with phosphoramidate linkag es to improve stability, are capable of degrading exogenous mRNA up to 27 hours after injection in the oocyte, For this reason, the phenotyp e could not be rescued by injection of a synthetic CK1/8 mRNA, However , modification of the synthetic CK1/8 mRNA, which prevents annealing o f the antisense oligonucleotide used for depleting the endogenous CK1/ 8 mRNA, did result in the rescue of the CK1/8 depletion phenotype. The se results demonstrate that the phenotype observed after depletion of the CK1/8 mRNA is truly caused by the lack of CK1/8 protein. Injection of the closely related type II cytokeratin (CK55) did not result in t he same level of rescue of the CK1/8 depletion phenotype, suggesting t hat structurally similar members of the cytokeratin family, expressed at different stages of development, cannot substitute for each other i n the early embryo.