Identification and characterization of proteins that selectively interact with the LHR mRNA binding protein (LRBP) in rat ovaries.
Luteinizing hormone receptor (LHR) mRNA binding protein (LRBP), identified as mevalonate kinase, has been shown to be a trans factor mediating the post-transcriptional regulation of LHR mRNA expression in ovaries. LRBP binds to the coding region of LHR mRNA and accelerates its degradation. Our previous studies in an in vitro system showed that LRBP represses the translation of LHR mRNA by forming an untranslatable ribonucleoprotein (mRNP) complex, further suggesting that the untranslatable mRNP complex is directed to the mRNA repression/decay machinery for subsequent mRNA turnover. In the present studies, we used yeast two-hybrid system to screen a cDNA library which was constructed from LHR down-regulated ovaries. Two proteins were identified interacting with LRBP: ribosomal protein S20 (RP S20) and ubiquitin conjugating enzyme 2i (UBCE2i). Their interactions with LRBP were confirmed by the mating assay, co-immunoprecipitation analyses and in vitro sumoylation assays. Furthermore, we show that LRBP is a target for modification by SUMO2/3 but not by SUMO1, at K256 and/or K345. Mutation of both lysine residues is sufficient to abrogate the sumoylation of LRBP. These findings suggest that the direct interaction of LRBP with the translation machinery, through RP S20, may be responsible for the transition of LHR mRNA to an untranslatable complex, and that sumoylation of LRBP may play a role in targeting the untranslatable mRNP complex to the mRNA decay machinery in specific cytoplasmic foci.
Wang
Lei
L
Department of Obstetrics and Gynecology, University of Michigan Medical School, Ann Arbor, MI-48109, USA.
Gulappa
Thippeswamy
T
Menon
K M J
KM
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010053:Ovary / Q000166:cytology / Q000378:metabolism*; D025941:Protein Interaction Mapping; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D009418:S100 Proteins / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*
C402897:LRBP-1 protein, rat; D016601:RNA-Binding Proteins; D009418:S100 Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes