Isolation and characterization of a novel trans-factor for luteinizing hormone receptor mRNA from ovary.
Post-transcriptional mechanisms play a major role in regulating luteinizing hormone (LH) receptor mRNA expression in the ovary. An ovarian cytosolic protein that we have identified in rats and humans, which binds to a polypyrimidine-rich bipartitate sequence in the coding region of LHR mRNA, acts as a trans-acting factor in this process. In the present study, we isolated and characterized this LH receptor mRNA-binding protein (LRBP) from rat ovary. LRBP was purified to homogeneity by cation exchange chromatography followed by Northwestern analysis and subsequent elution of the single protein band from SDS-polyacrylamide gel. Purified LRBP was subjected to N-terminal microsequencing followed by homology search, which revealed its identity as mevalonate kinase. Purified rat mevalonate kinase antibody recognized the gel-purified LRBP on Western blots performed with one- and two-dimensional SDS-polyacrylamide gels. When recombinant mevalonate kinase produced in human embryonic kidney cells (293 cells) was tested, it showed all of the characteristics of LRBP with respect to specificity of LHR mRNA binding sequence, as examined by gel mobility shift analysis. Inhibition of LHR mRNA binding activity of mevalonate kinase in the presence of ATP and mevalonate indicates that the RNA recognition site of mevalonate kinase might involve the ATP/mevalonate binding region of the protein. Treatment of 293 cells with mevastatin to deplete cellular mevalonate resulted in an increase in LHR mRNA binding activity of mevalonate kinase. Collectively, the data support the novel function of rat mevalonate kinase as a LHR mRNA-binding protein in the post-transcriptional regulation of LH receptor expression in the ovary.
Nair
Anil K
AK
Departments of Biological Chemistry and Obstetrics/Gynecology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0617, USA.
Menon
K M J
KM
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002412:Cations; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D010053:Ovary / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011974:Receptors, LH / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
D002412:Cations; C402897:LRBP-1 protein, rat; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011974:Receptors, LH; D012313:RNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase