Identification of a novel mRNA-associated protein in oocytes of Pleurodeles waltl and Xenopus laevis.
Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the complete Pleurodeles cDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS-PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)+ RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein-protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinant Pleurodeles RAP55 fragment recognize the protein from Pleurodeles and Xenopus. The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.
Lieb
B
B
Biocenter, University of Würzburg, Am Hubland, Würzburg, D-97074, Germany. lieb@pop.uni-mainz.de
Carl
M
M
Hock
R
R
Gebauer
D
D
Scheer
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004527:Egg Proteins; D005260:Female; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism; D009928:Organ Specificity; D010999:Pleurodeles / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology / Q000378:metabolism*
D004527:Egg Proteins; C116494:RAP55 protein, Pleurodeles waltl; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D012260:Ribonucleases