Modified mRNA rescue of maternal CK1/8 mRNA depletion in Xenopus oocytes.
This work addresses two issues, the use of antisense oligodeoxynucleotides to deplete specific mRNAs in Xenopus oocytes to analyze their functions during development and the role of cytokeratin filaments in cells of the early Xenopus embryo. We have shown previously that depletion of cytokeratin CK1/8 mRNA causes defects in the early embryo. In this study, we show that the oligos, modified with phosphoramidate linkages to improve stability, are capable of degrading exogenous mRNA up to 27 hours after injection in the oocyte. For this reason, the phenotype could not be rescued by injection of a synthetic CK1/8 mRNA. However, modification of the synthetic CK1/8 mRNA, which prevents annealing of the antisense oligonucleotide used for depleting the endogenous CK1/8 mRNA, did result in the rescue of the CK1/8 depletion phenotype. These 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 the same level of rescue of the CK1/8 depletion phenotype, suggesting that structurally similar members of the cytokeratin family, expressed at different stages of development, cannot substitute for each other in the early embryo.
Raats
J M
JM
Department of Biochemistry, University of Nijmegen, The Netherlands.
Gell
D
D
Vickers
L
L
Heasman
J
J
Wylie
C
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001757:Blastomeres / Q000187:drug effects / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000502:physiology*; D005260:Female; D018392:Genomic Imprinting; D007633:Keratins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D007633:Keratins