RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway.
Oocyte meiosis and early mitotic divisions in developing embryos rely on the timely production of cell cycle regulators and their clearance via proteasomal degradation. Ret Finger Protein-Like 4 (Rfpl4), encoding a RING finger-like protein with a B30.2 domain, was discovered during an in silico search for germ cell-specific genes. To study the expression and functions of RFPL4 protein, we performed immunolocalizations and used yeast two-hybrid and other protein-protein interaction assays. Immunohistochemistry and immunofluorescence showed that RFPL4 accumulates in all growing oocytes and quickly disappears during early embryonic cleavage. We used a yeast two-hybrid model to demonstrate that RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, proteasome subunit beta type 1, ubiquitin B, as well as a degradation target protein, cyclin B1. Coimmunoprecipitation analyses of in vitro translated proteins and extracts of transiently cotransfected Chinese hamster ovary (CHO)-K1 cells confirmed these findings. We conclude that, like many RING-finger containing proteins, RFPL4 is an E3 ubiquitin ligase. The specificity of its expression and these interactions suggest that RFPL4 targets cyclin B1 for proteasomal degradation, a key aspect of oocyte cell cycle control during meiosis and the crucial oocyte-to-embryo transition to mitosis.
Suzumori
Nobuhiro
N
Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.
Burns
Kathleen H
KH
Yan
Wei
W
Matzuk
Martin M
MM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000502:physiology*; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D015723:Gene Library; D008025:Ligases / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D008540:Meiosis; D051379:Mice; D008810:Mice, Inbred C57BL; D009097:Multienzyme Complexes / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D009097:Multienzyme Complexes; D025801:Ubiquitin; C461681:Rfpl4 protein, mouse; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases