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RFPL4 Interacts with Oocyte Proteins of the Ubiquitin-Proteasome Degradation Pathway

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RFPL4 Interacts with Oocyte Proteins of the Ubiquitin-Proteasome Degradation Pathway RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway Nobuhiro Suzumori*†, Kathleen H. Burns*‡, Wei Yan*, and Martin M. Matzuk*‡§¶ Departments of *Pathology, ‡Molecular and Human Genetics, and §Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and †Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya 467-0001, Japan Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved November 25, 2002 (received for review July 26, 2002) Oocyte meiosis and early mitotic divisions in developing embryos Interestingly, there are recurring biological themes in the rely on the timely production of cell cycle regulators and their regulation of protein translation and protein clearance during clearance via proteasomal degradation. Ret Finger Protein-Like 4 meiosis that are conserved between species and between sexes. (Rfpl4), encoding a RING finger-like protein with a B30.2 domain, We are beginning to appreciate the central roles of ubiquitin- was discovered during an in silico search for germ cell-specific mediated proteasomal degradation pathways in these processes genes. To study the expression and functions of RFPL4 protein, we (7–9). Mutations in the human gene ubiquitin-specific protease performed immunolocalizations and used yeast two-hybrid and 9 Y chromosome (USP9Y), which encodes a protein with a other protein–protein interaction assays. Immunohistochemistry C-terminal ubiquitin hydrolase domain, result in azoospermia and immunofluorescence showed that RFPL4 accumulates in all and male infertility (10). Knockout mice lacking the E3 ubiquitin growing oocytes and quickly disappears during early embryonic protein ligase SIAH1A or the E2 ubiquitin-conjugating enzyme cleavage. We used a yeast two-hybrid model to demonstrate that HR6B demonstrate defects in meiosis and postmeiotic germ-cell RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, development and male infertility (11, 12). Ubiquitin-mediated proteasome subunit ␤ type 1, ubiquitin B, as well as a degradation proteolysis is also critical for other aspects of reproduction, target protein, cyclin B1. Coimmunoprecipitation analyses of in including the elimination of defective sperm in the epididymis, vitro translated proteins and extracts of transiently cotransfected clearance of paternal mitochondria, and progression of embry- Chinese hamster ovary (CHO)-K1 cells confirmed these findings. We onic development in mammals, as well as degradation of the conclude that, like many RING-finger containing proteins, RFPL4 is vitelline coat during fertilization in ascidians (13–16). an E3 ubiquitin ligase. The specificity of its expression and these Maturation-promoting factor (MPF), a heterodimer of interactions suggest that RFPL4 targets cyclin B1 for proteasomal p34Cdc2 kinase and cyclin B1, is a key regulator of oocyte degradation, a key aspect of oocyte cell cycle control during meiosis that functions during discrete periods of the cell cycle. meiosis and the crucial oocyte-to-embryo transition to mitosis. MPF activity increases during prophase and metaphase of meiosis I because of increased translation of cyclin B1 mRNAs proteolysis ͉ meiotic regulator ͉ maturation promoting factor and the dephosphorylation of p34Cdc2 in complexes with cyclin B1. As oocytes progress through meiosis I, MPF is transiently inactivated by the proteasomal degradation of cyclin B1 (17, 18). biquitination is the major means in eukaryotic cells for Subsequently, translation of several key oocyte mRNAs (e.g., targeted protein proteolysis (1). By covalent addition of U cyclin B1 and Mos) and activation of Cdc2 kinase by Cdc25 polyubiquitin to specific proteins, the ubiquitination system phosphatase occur, so that MPF activity is high in metaphase II. regulates protein levels and thereby influences diverse cellular Egg–sperm fusion at fertilization releases the oocyte from processes. There are three well established types of enzymes ϩ ϩ metaphase II arrest by increasing Ca2 levels, activating Ca2 - involved in ubiquitination, termed E1, E2, and E3. E1 is the calmodulin kinase II, and targeting cyclin B1 and MOS for ubiquitin-activating enzyme, which forms a thiol-ester linkage degradation via the ubiquitin proteasome pathway (19–21). with ubiquitin through its active site cysteine. Ubiquitin is These studies indicate that specific ubiquitination pathways subsequently transferred to an E2 ubiquitin-conjugating enzyme. regulate MPF at several key transitions in oocyte meiosis. The E3 enzyme is the ubiquitin protein ligase, which transfers Here, we describe the expression of RFPL4 and its protein- ubiquitin from the E2 enzyme to lysines of a specific protein, protein interactions. Together, these findings suggest that targeting the protein for degradation by the proteasome. More RFPL4 functions as an E3 ubiquitin protein ligase to regulate recently, E4 enzymes have been described that appear to func- protein degradation and meiotic cell cycle progression in mouse tion in ubiquitin chain polymerization (2). Few E1 enzymes, oocytes. several E2 enzymes, and hundreds of E3 enzymes have been identified. It is the E3 ubiquitin protein ligase that adds speci- Materials and Methods ficity to the process by interacting with specific target proteins. Generation of the Anti-RFPL4 Antibody and RFPL4 Protein Analysis. Included in the group of E3 enzymes are proteins such as the A full-length Rfpl4 cDNA fragment was subcloned into pET-23b cancer-associated proteins, anaphase-promoting complex (Novagen), His-tagged RFPL4 was produced in BL21 [DE3] (APC), BRCA1, and MDM2, and the DNA repair proteins, pLysS cells, and polyclonal antibodies were raised in goats RAD5 and RAD18. Many E3 ubiquitin protein ligases share a (Cocalico Biologicals, Reamstown, PA). Immunofluorescence, common RING (Really Interesting Novel Gene) finger con- ͞ Western blot, and immunohistochemical analysis were per- sensus sequence CX2CX (9–39)CX (1–3)HX (2–3)C HX2CX formed as described (22, 23). Before immunofluorescence, fixed (4–48)CX2C (reviewed in refs. 3 and 4), and a majority of RING oocytes and embryos were permeabilized for 20 min in PBS finger-containing proteins have been shown to function as E3 with 10% FCS and 0.2% Triton X-100. These were imaged ubiquitin protein ligases. by deconvolution microscopy (Axiovert S100 2TV, Zeiss, Ger- A few related proteins have been identified and termed Ret Finger Protein-Like 1 (RFPL1), RFPL2, and RFPL3. These RFPL proteins are unique members of the RING finger- This paper was submitted directly (Track II) to the PNAS office. containing family that do not have a histidine in their RING Abbreviations: CHO, Chinese hamster ovary; MPF, maturation-promoting factor; APC, finger motif (5). We recently identified a fourth RFPL member, anaphase-promoting complex; GV, germinal vesicle. RFPL4, expressed specifically in germ cells (6). ¶To whom correspondence should be addressed. E-mail: [email protected]. 550–555 ͉ PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0234474100 Downloaded by guest on September 25, 2021 many). For Western blot analysis, total protein from 150 oocytes or embryos, or 20 ␮g of tissue protein extract were loaded in each lane. cDNA Library Construction. Ovaries were collected from adult C57BL͞6J͞129͞SvEv hybrid Gdf9 knockout (Gdf9Ϫ/Ϫ) mice (24) (6–16 weeks of age) for RNA isolation by using the RNA STAT-60 reagent (Leedo Medical Laboratories, Houston). To collect germinal vesicle (GV)-stage oocytes, (C57BL͞6J ϫ SJL͞J) F1 mice were killed 48 h after pregnant mare serum gonadotropin (PMSG) treatment to stimulate follicle develop- ment. Oocytes were denuded by pipetting, and mRNA was extracted by using the MicroFast Track 2.0 kit (Invitrogen). RNA was reverse-transcribed, and cDNAs were size selected and subcloned into SmaI-linearized pGADT7-Rec cloning vector (CLONTECH). Yeast Two-Hybrid Screen and Construction of Truncation Constructs. A yeast two-hybrid screen (MATCHMAKER, CLONTECH) Rfpl4 Fig. 1. Western blot analysis of RFPL4 expression. Anti-RFPL4 goat polyclonal was performed by using pGBKT7-full-length mouse cDNA Ϫ/Ϫ (GenBank accession no. AY070253) as bait. After yeast mating, antibodies detect RFPL4 protein in lysates from wild-type (Wt) and Gdf9 ͞ ͞ ͞ ͞ ␣ ovary samples and early embryos, but not from heart (He), liver (Li), spleen clones that grew on (Leu- Trp- Ade- His- X- -Gal) selection (Sp), or testes (Te). No extraneous bands were detected. RFPL4 protein is plates were isolated, and candidate pGADT7-cDNAs were present in unfertilized GV stage oocytes (Oo), and two-cell (2C) embryos, but sequenced. For cotransformation truncation constructs, por- not in eight-cell (8C) embryos. Actin in the tissue lysates is shown as a control tions of Rfpl4 and cyclin B1 (Ccnb1, BC011478) were used (Fig. for protein loading. RFPL4 recombinant protein was not detected by using 2A); expression vectors containing full-length cyclin B1 proved preimmune serum samples from the same goat (data not shown). toxic to yeast. The full-length cDNA encoding HR6A (Ube2a, AF383148), was also subcloned into yeast expression vector Ϫ Ϫ for these analyses. All constructs were confirmed by DNA ovary, and Gdf9 / ovary (enriched in oocytes) were used. In sequencing. addition, oocytes, two-cell embryos, and eight-cell embryos were collected from the ovaries or oviducts of wild-type female mice. In Vitro Transcription͞Translation and Coimmunoprecipitation. The The RFPL4 antiserum
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