Polyadenylation-mediated translational regulation of maternal P450(11beta) mRNA in frog oocytes.
Northern blot analysis of bullfrog tissues using a cDNA probe of cytochrome P450(11beta) showed that a large amount of message was present in the ovary as well as in the adrenal tissue. Two kinds of mRNA of different sizes were found in the ovary. Sequence determination of the two cDNAs and analysis by reverse-transcription polymerase chain reaction indicated that the protein encoded by the larger mRNA was identical to the adrenal enzyme, while the protein encoded by the smaller had a truncated sequence lacking an extension peptide necessary for the protein transport to the mitochondria. The mRNAs were present in the oocytes but not in the follicular cells, and their content in an oocyte varied little during its maturation. Immunoblot analyses of the mitochondrial fraction of oocytes failed to demonstrate the presence of P450(11beta) protein. In contrast the eggs were found to contain a large amount of enzymatically active protein. Interestingly the mRNA has a cis-element called cytoplasmic polyadenylation element at its 3' untranslated region. When poly(A) tails of the message prepared from eggs and oocytes were examined by RNase H digestion or reverse-transcription polymerase chain reaction, those of eggs were about 150 nucleotides longer than those of oocytes. These results suggest that translation of the message is stimulated during the oocyte maturation as a result of enhanced polyadenylation at its 3'-end. Finally a finding is presented that progesterone was converted to 11beta-hydroxyprogesterone by the frog P450(11beta), implying that the enzyme expressed in eggs may control a level of progesterone which is needed to initiate the oocyte maturation.
Takemori
H
H
Department of Molecular Physiological Chemistry, Osaka University Medical School, Suita, Japan.
Halder
S K
SK
Nonaka
Y
Y
Fujii
T
T
Ohta
M
M
Hatano
O
O
Okamoto
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006908:Hydroxyprogesterones / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology* / Q000378:metabolism; D009866:Oogenesis; D011374:Progesterone / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011898:Ranidae; D016914:Ribonuclease H / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics*
D006908:Hydroxyprogesterones; D012333:RNA, Messenger; C030228:11-hydroxyprogesterone; D011374:Progesterone; D013252:Steroid 11-beta-Hydroxylase; D016914:Ribonuclease H