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 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  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. proteolysis 兩 meiotic regulator 兩 maturation promoting factor
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biquitination is the major means in eukaryotic cells for targeted protein proteolysis (1). By covalent addition of polyubiquitin to specific proteins, the ubiquitination system regulates protein levels and thereby influences diverse cellular processes. There are three well established types of enzymes involved in ubiquitination, termed E1, E2, and E3. E1 is the ubiquitin-activating enzyme, which forms a thiol-ester linkage with ubiquitin through its active site cysteine. Ubiquitin is subsequently transferred to an E2 ubiquitin-conjugating enzyme. The E3 enzyme is the ubiquitin protein ligase, which transfers ubiquitin from the E2 enzyme to lysines of a specific protein, targeting the protein for degradation by the proteasome. More recently, E4 enzymes have been described that appear to function in ubiquitin chain polymerization (2). Few E1 enzymes, several E2 enzymes, and hundreds of E3 enzymes have been identified. It is the E3 ubiquitin protein ligase that adds specificity to the process by interacting with specific target proteins. Included in the group of E3 enzymes are proteins such as the cancer-associated proteins, anaphase-promoting complex (APC), BRCA1, and MDM2, and the DNA repair proteins, RAD5 and RAD18. Many E3 ubiquitin protein ligases share a common RING (Really Interesting Novel Gene) finger consensus sequence CX2CX (9 –39)CX (1–3)HX (2–3)C兾HX2CX (4 – 48)CX2C (reviewed in refs. 3 and 4), and a majority of RING finger-containing proteins have been shown to function as E3 ubiquitin protein ligases. 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 fingercontaining family that do not have a histidine in their RING finger motif (5). We recently identified a fourth RFPL member, RFPL4, expressed specifically in germ cells (6). 550 –555 兩 PNAS 兩 January 21, 2003 兩 vol. 100 兩 no. 2
Interestingly, there are recurring biological themes in the regulation of protein translation and protein clearance during meiosis that are conserved between species and between sexes. We are beginning to appreciate the central roles of ubiquitinmediated proteasomal degradation pathways in these processes (7–9). Mutations in the human gene ubiquitin-specific protease 9 Y chromosome (USP9Y), which encodes a protein with a C-terminal ubiquitin hydrolase domain, result in azoospermia and male infertility (10). Knockout mice lacking the E3 ubiquitin protein ligase SIAH1A or the E2 ubiquitin-conjugating enzyme HR6B demonstrate defects in meiosis and postmeiotic germ-cell development and male infertility (11, 12). Ubiquitin-mediated proteolysis is also critical for other aspects of reproduction, including the elimination of defective sperm in the epididymis, clearance of paternal mitochondria, and progression of embryonic development in mammals, as well as degradation of the vitelline coat during fertilization in ascidians (13–16). Maturation-promoting factor (MPF), a heterodimer of p34Cdc2 kinase and cyclin B1, is a key regulator of oocyte meiosis that functions during discrete periods of the cell cycle. MPF activity increases during prophase and metaphase of meiosis I because of increased translation of cyclin B1 mRNAs 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). Subsequently, translation of several key oocyte mRNAs (e.g., cyclin B1 and Mos) and activation of Cdc2 kinase by Cdc25 phosphatase occur, so that MPF activity is high in metaphase II. Egg–sperm fusion at fertilization releases the oocyte from metaphase II arrest by increasing Ca2⫹ levels, activating Ca2⫹calmodulin kinase II, and targeting cyclin B1 and MOS for degradation via the ubiquitin proteasome pathway (19–21). These studies indicate that specific ubiquitination pathways regulate MPF at several key transitions in oocyte meiosis. Here, we describe the expression of RFPL4 and its proteinprotein interactions. Together, these findings suggest that RFPL4 functions as an E3 ubiquitin protein ligase to regulate protein degradation and meiotic cell cycle progression in mouse oocytes. Materials and Methods Generation of the Anti-RFPL4 Antibody and RFPL4 Protein Analysis.
A full-length Rfpl4 cDNA fragment was subcloned into pET-23b (Novagen), His-tagged RFPL4 was produced in BL21 [DE3] pLysS cells, and polyclonal antibodies were raised in goats (Cocalico Biologicals, Reamstown, PA). Immunofluorescence, Western blot, and immunohistochemical analysis were performed as described (22, 23). Before immunofluorescence, fixed oocytes and embryos were permeabilized for 20 min in PBS with 10% FCS and 0.2% Triton X-100. These were imaged by deconvolution microscopy (Axiovert S100 2TV, Zeiss, GerThis paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CHO, Chinese hamster ovary; MPF, maturation-promoting factor; APC, anaphase-promoting complex; GV, germinal vesicle. ¶To
whom correspondence should be addressed. E-mail:
[email protected].
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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 C57BL兾6J兾129兾SvEv hybrid Gdf9 knockout (Gdf9⫺/⫺) 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, (C57BL兾6J ⫻ SJL兾J) F1 mice were killed 48 h after pregnant mare serum gonadotropin (PMSG) treatment to stimulate follicle development. 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.
In Vitro Transcription兾Translation and Coimmunoprecipitation. The
pGBKT7 (MYC-tagged) and pGADT7 (hemagglutinin-tagged) vectors were used as templates for in vitro transcription兾 translation using [35S]Met and the TNT T7 Coupled Reticulocyte Lysate System (Promega). In vitro translated proteins were combined at room temperature for 1 h, and reciprocal coimmunoprecipitation experiments were performed by using mouse anti-MYC monoclonal or rabbit anti-hemagglutinin polyclonal antibodies (CLONTECH).
Cell Culture, Plasmids, and Transfection. Chinese hamster ovary (CHO)-K1 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium兾 Ham’s F-12 (DMEM兾F-12) containing 10% FBS and grown to 90–95% confluence in 6-cm dishes. To express tagged proteins, mouse cDNAs were inserted into pCMV-Tag4A兾FLAG-C and pCMV-Tag5A兾MYC-C vectors (Stratagene) and transiently transfected by using LipofectAMINE 2000 (Invitrogen). Twentyfour hours after transfection, cells were harvested, processed in lysis buffer [50 mM Tris䡠HCl, pH 7.4兾150 mM NaCl兾1 mM EDTA兾1% Triton X-100兾protease inhibitor mixture (Sigma)], and analyzed by immunoprecipitation and SDS兾PAGE. Cell Extract Immunoprecipitation and Western Blot Analysis. Immu-
noprecipitations were performed by using the FLAG-Tagged Protein Immunoprecipitation kit (Sigma) as described by the manufacturer. The bound antibody was detected by the ECL detection kit (Amersham Pharmacia). Results
Production of RFPL4 Protein and Specificity of the Anti-RFPL4 Antibody. We produced recombinant full-length mouse RFPL4 pro-
tein in Escherichia coli and isolated the product by using His tag affinity. This reagent was used to generate polyclonal goat antibodies. Affinity and specificity of the anti-RFPL4 antibody were tested by Western blot analysis. Total protein extracts prepared from adult mouse wild-type heart, liver, spleen, testis, Suzumori et al.
Fig. 1. Western blot analysis of RFPL4 expression. Anti-RFPL4 goat polyclonal 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 (Sp), or testes (Te). No extraneous bands were detected. RFPL4 protein is present in unfertilized GV stage oocytes (Oo), and two-cell (2C) embryos, but not in eight-cell (8C) embryos. Actin in the tissue lysates is shown as a control for protein loading. RFPL4 recombinant protein was not detected by using preimmune serum samples from the same goat (data not shown).
ovary, and Gdf9⫺/⫺ ovary (enriched in oocytes) were used. In addition, oocytes, two-cell embryos, and eight-cell embryos were collected from the ovaries or oviducts of wild-type female mice. The RFPL4 antiserum detects RFPL4 in ovaries, oocytes, and two-cell embryos (Fig. 1) but fails to detect a band in other samples. This finding is consistent with our Northern blot analysis showing that Rfpl4 mRNA is ovary-specific (6). RFPL4 Protein Is Expressed in Growing Oocytes and Early Embryos.
RFPL4 protein is located in growing oocytes in both wild-type and Gdf9⫺/⫺ ovaries, beginning at the primary follicle stage and extending through the preovulatory follicle stage (Fig. 2 A–C). Deconvolution microscopy was used to assess RFPL4 protein expression in oocytes throughout meiotic maturation and in early preimplantation embryos. Fully grown oocytes were evaluated before the resumption of meiosis (GV stage) (Fig. 2D) and after ovulation and progression to metaphase II (Fig. 2G). In GV stage oocytes, RFPL4 protein is located in the cytoplasm and nucleus, and seems relatively excluded from the nucleolus (Fig. 2D); after nuclear membrane breakdown, RFPL4 is detected throughout the oocyte (Fig. 2G). No expression was discernable in oocyte-associated granulosa cells (data not shown). In addition, the RFPL4 protein is degraded in two-cell stage and eight-cell stage embryos (Fig. 2 E and F). Expression of RFPL4 was quantified by comparing immunofluorescent signal intensities in GV stage oocytes, metaphase II oocytes, and two-, four-, and eight-cell embryos (Fig. 3). These data strongly suggest a specific role for RFPL4 in oogenesis and oocyte meiosis. Yeast Two-Hybrid Screening of Mouse cDNA Libraries. The fulllength ORF of mouse Rfpl4 (Fig. 4A) corresponding to amino acid residues 1–287 was subcloned into the pGBKT7 vector for expression as a GAL4 DNA binding fusion protein. Ovarian and oocyte cDNA libraries were subcloned into the pGADT7 vector to be expressed as transactivation domain fusion proteins. In this yeast two-hybrid system, interactions between RFPL4 and proteins encoded by library cDNAs are expected to reconstitute transactivating complexes, which bind to DNA and promote transcription of selectable markers. To identify RFPL4interacting proteins, we screened ⬇1 ⫻ 106 ovary cDNA transPNAS 兩 January 21, 2003 兩 vol. 100 兩 no. 2 兩 551
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A yeast two-hybrid screen (MATCHMAKER, CLONTECH) was performed by using pGBKT7-full-length mouse Rfpl4 cDNA (GenBank accession no. AY070253) as bait. After yeast mating, clones that grew on (Leu-兾Trp-兾Ade-兾His-兾X-␣-Gal) selection plates were isolated, and candidate pGADT7-cDNAs were sequenced. For cotransformation truncation constructs, portions of Rfpl4 and cyclin B1 (Ccnb1, BC011478) were used (Fig. 2 A); expression vectors containing full-length cyclin B1 proved toxic to yeast. The full-length cDNA encoding HR6A (Ube2a, AF383148), was also subcloned into yeast expression vector for these analyses. All constructs were confirmed by DNA sequencing.
Fig. 2. Immunohistochemical analysis of RFPL4 in mouse ovaries, oocytes, and embryos. Seven-week-old wild-type (A and B) and Gdf9 knockout (C) ovary photographed under low (A and B) or high (C) magnification. In the adult wild-type ovary (A and B), RFPL4 immunoreactivity (red) was detected in oocytes of primary (1F), secondary (2F), and antral follicles (AF). Oocytes in AF of the wild-type mouse ovary are heavily stained (B). Folliculogenesis is blocked at primary follicle stage in Gdf9 knockout mice (24). (C) In the Gdf9 knockout mouse ovary, RFPL4 immunoreactivity is detected in oocytes of primary follicles. (D) RFPL4 protein can be detected by immunofluorescence (red) in oocytes preceding the resumption of meiosis. Immunofluorescence in GV stage oocytes both with (D) and without (data not shown) their zona pellucida demonstrated a relative exclusion of RFPL4 antigenicity from the nucleolus (N). RFPL4 is rapidly degraded in early preimplantation embryos between the two-cell (2c) (E) and eight-cell (8c) (F) stage. (G) Shows two unfertilized oocytes arrested in metaphase II (MII) (bright red) next to a blastocyst (BI) with no detectable RFPL4 protein. Preimmune goat serum was used as a negative control; a GV stage oocyte, surrounded by granulosa cells, is shown before resumption of meiosis (BL) (H).
formants by mating. A total of ⬎600 colonies grew on Leu-兾 Trp-兾Ade-兾His-兾X-␣-Gal selection plates, and 27 of the isolated plasmids with inserts ⬎500 bp were sequenced. Seven of these sequences were in-frame portions of the proteasome subunit , type 1 (PSMB1) coding sequence (U60824). We also screened ⬇1 ⫻ 106 oocyte cDNA transformants by using the pGBKT7-RFPL4 bait. A total of ⬎200 colonies grew on the stringent selection plates, and eight pGADT7 plasmids with large inserts were sequenced. One sequence corresponded to ubiquitin B cDNA (Ubb NM㛭011664). One of the transfor-
mant cDNAs corresponded to PSMB1. Other sequences identified in these screens are provided in Table 1, which is published as supporting information on the PNAS web site, www.pnas.org.
Fig. 3. Quantitative analysis of RFPL4 immunofluorescence in oocytes and early embryos. High levels of RFPL4 are detected in GV stage oocytes (GV) and metaphase II (MII) oocytes. RFPL4 signal is diminished in two-cell (2c) and four-cell (4c) embryos, and is not detected in eight-cell (8c) morula. Average intensities after 0.5-s exposures are given with prebleed intensities subtracted. Error bars represent the SEM of each sample (n ⫽ 10).
Fig. 4. Truncation constructs of RFPL4 and cyclin B1. (A) Schematic representation of RFPL4, and ⌬C79, ⌬N86, ⌬N79⌬C155, and ⌬N79 derivatives. The RING finger-like region and B30.2 domain are shown as gray and black boxes, respectively. (B) Schematic representation of cyclin B1, and ⌬C198, and ⌬N251 derivatives. The destruction box (D-box) and cyclin box are shown as gray and black boxes, respectively.
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RFPL4-Interacting Proteins in Yeast. Based on the above protein–
protein interactions and the primary structure of RFPL4 (Fig.
Suzumori et al.
Fig. 5. The strength of protein–protein interactions assessed by cotransformant and yeast mating fluorescence assays. After inoculation, we checked the initial fluorescence, allowed the cells to grow for 40 h, and then rechecked fluorescence of an oxygen sensitive dye by using the MATCHMAKER Biosensor kit (CLONTECH). Fluorescence was excited at 485 nm, and emission was read at 630 nm. To find the fold increase in fluorescence, an indication of yeast growth in selection media, we divided the fluorescence intensity at time t ⫽ 40 h by the initial intensity recorded at time t ⫽ 0 h [Fluorescence(t40)兾 Fluorescence(t0)]. The values shown are the means of triplicate measurements of three independent transformants. Standard deviations are indicated by the error bars. The pGBKT7-murine p53 (p53) and pGADT7-SV40 large T-antigen (Large T) were used for positive control. (A) Cotransformant assay. (B and C) Yeast mating assay.
4A), we proposed that RFPL4 could act as an E3 ubiquitin ligase to transfer ubiquitin from the HR6A E2 conjugating enzyme to targets such as cyclin B1. To test interactions between RFPL4 and HR6A, cyclin B1, PSMB1, or UbB in yeast, we cotransformed pGBKT7-RFPL4 and pGADT7 constructs and assessed growth with Leu-兾Trp-兾Ade-兾His-兾X-␣-Gal selection. To assess the strength of each interaction, we used a fluorometric method for measuring yeast growth after cotransformation and mating. We considered that strong protein–protein interactions would result in a 3-fold increase in dye fluorescence over a 40-h incubation period. Cotransformants of pGBKT7-murine p53 and pGADT7-SV40 large T antigen were used as a positive control (⬎5-fold increase), and cotransformants of pGBKT7RFPL4 and empty pGADT7 vector were used as a negative control (⬇1-fold). We found rapid growth of cotransformants with pGBKT7-RFPL4 and pGADT7-HR6A, -N-terminal cyclin B1 (CCNB1⌬C198; Fig. 4B), -PSMB1, or -UbB, but not the cyclin B1 C terminus (CCNB1⌬N251) (Fig. 5A and data not shown). In contrast, yeast growth assays suggest that HR6A interacts strongly with the C terminus of cyclin Suzumori et al.
In Vitro Protein Interactions. To confirm the validity of our yeast two-hybrid results, we performed coimmunoprecipitations of proteins expressed in vitro in a rabbit reticulocyte lysate system. Epitope-tagged bait and prey proteins were transcribed by T7 polymerase from pGBKT7 and pGADT7 templates without DNA binding or transactivation domains. As positive control vectors, we used pGBKT7-murine p53 (MYC epitope-tagged) and pGADT7-SV40 large T (hemagglutinin epitope-tagged). [35S]methionine was included in translation mixtures to generate products detectable by autoradiography. RFPL4 (32 kDa) interacts with HR6A (17 kDa) and fulllength cyclin B1 (47 kDa) and HR6A binds full length cyclin B1 (Fig. 8, which is published as supporting information on the PNAS web site). Interactions between UbB (33 kDa) and RFPL4 (32 kDa) were not assessed because the two proteins could not be readily resolved by electrophoresis under these conditions. Other putative RFPL4 interactions identified in the yeast two hybrid screen were not verifiable by cotransformant assays and coimmunoprecipitation approaches (Table 1). Protein Interactions in CHO Cells. To confirm that RFPL4 binds to
HR6A, cyclin B1, and PSMB1 in mammalian cells, coimmunoprecipitation studies were performed by using extracts of transiently transfected CHO cells. The interaction between HR6A (17 kDa) and cyclin B1 (47 kDa) was also studied by this approach. Anti-FLAG antibodies could coimmunoprecipitate FLAG-tagged, full-length RFPL4 bound to MYC-tagged HR6A, cyclin B1, or PSMB1 (25 kDa) from lysates of CHO cells cotransfected with these constructs (Fig. 6A). Likewise, the anti-FLAG antibodies could coimmunoprecipitate FLAGtagged cyclin B1 and MYC-tagged HR6A or FLAG-tagged cyclin B1 and MYC-tagged RFPL4 (Fig. 6B). No deleterious effects were observed in any transiently transfected cells after 48 h of expression. Because the RFPL4 B30.2 domain interacts with cyclin B1 and HR6A in vitro, we expressed a truncated form of RFPL4 that lacks the RING finger-like motif (RFPL4⌬N79), along with cyclin B1 or HR6A; however, binding was not demonstrated in CHO cells (data not shown). In addition, we PNAS 兩 January 21, 2003 兩 vol. 100 兩 no. 2 兩 553
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B1 (CCNB1⌬N251), but not the N terminus of cyclin B1 (CCNB1⌬C198) (Fig. 5B). In addition, there was no growth of cotransformants with pGBKT7-RFPL4 and pGADT7-RFPL4 (data not shown), suggesting that RFPL4 did not interact with itself in yeast. RFPL4 has a tripartite structure consisting of a cysteine-rich (C3YC4) RING finger-like region, a coiled-coiled motif, and a B30.2 domain, characteristics of RING-B30 family proteins (6). To determine regions of RFPL4 that mediate its interactions, we engineered a series of truncation mutants (Fig. 4A). The Cterminal B30.2 domain (amino acids 79–287; RFPL4⌬N79) proved both necessary and sufficient to recreate all of the interactions studied (Fig. 5 B and C and data not shown), demonstrating that the RING finger-like region is dispensable for these strong interactions with HR6A and the N terminus of cyclin B1. Deletion of the first 154 aa of RFPL4 (the RING finger-like region and a portion of the B30.2 domain; RFPL4⌬N155) weakened the interactions slightly with HR6A and CCNB1⌬C198 (Fig. 5 B and C), but did not prevent the binding to UbB (data not shown). Deletion of all of the B30.2 domain of RFPL4 (RFPL4⌬C79) or a C-terminal portion (RFPL4⌬N79⌬C155 and RFPL4⌬C155) abolished or significantly weakened, respectively, the interactions with HR6A (Fig. 5B). The RFPL4 B30.2 domain (RFPL4⌬N79 or RFPL4⌬N155) also interacts with the N terminus of cyclin B1 (CCNB1⌬C198) (Fig. 5C). The RFPL4-cyclin B1 interaction data support the findings that cyclin B1 ubiquitination depends on a destruction box (D-box) sequence located in the N terminus of cyclin B1 (25).
Fig. 6. Protein–protein interactions in CHO cells. (A) Immunoprecipitation (IP) was performed with anti-FLAG antibodies. By Western blot analysis, MYC-tagged constructs were detected with anti-MYC antibodies (Upper); FLAG-tagged constructs were detected with anti-FLAG antibodies (Lower) as a loading control. The MYC-tagged HR6A, cyclin B1, and PSMB1 are detected in the immunoprecipitate with FLAG-tagged RFPL4. (B) MYC-tagged HR6A and RFPL4 were immunoprecipitated in association with FLAG-tagged cyclin B1. In these Western blots, FLAG-tagged RFPL4 and cyclin B1 constructs were detected with anti-FLAG antibodies (1:1,000 dilution), and MYC-tagged constructs were detected with anti-MYC antibodies (1:1,000 dilution).
examined MYC-tagged UbB coexpressed with FLAG-tagged RFPL4 in CHO cells, but could not detect an interaction. These latter findings imply that these interactions are weak in CHO cells, possibly because of the presence of other factors that regulate the binding of these proteins. Discussion Our laboratory recently reported the discovery of the Rfpl4 gene (6), which we now demonstrate encodes an oocyte-specific RING finger-like protein. RFPL4 protein accumulates to high levels during oogenesis and is rapidly lost in early cleavage stage embryos. Based on the primary amino acid sequence, we speculated that RFPL4 may function as an E3 ubiquitin ligase to target proteins for proteasomal degradation. A yeast two-hybrid screen and immunoprecipitation studies conducted with in vitro translated protein and transiently transfected CHO cell protein extracts provide compelling evidence to support this hypothesis. We found that RFPL4 associates with the E2 conjugating enzyme HR6A and the N-terminal D-box motif of cyclin B1, a target of ubiquitin-mediated proteasomal degradation during meiosis (26). Thus, RFPL4 forms a physical connection between an upstream enzyme that prepares ubiquitin for attachment to target proteins and cyclin B1. In addition, RFPL4 interacts with the proteasomal subunit PSMB1 in vitro and may tether targeted cyclin B1 to the proteasome or directly participate in cyclin B1 destruction. On completion of its roles in oocytes and early embryos, RFPL4 itself may be targeted for proteolysis by related degradation pathways. This would be consistent with the rapid cessation of RFPL4 protein expression in preimplantation embryos as well as the protein–protein interactions that we have discovered. Meiosis is a highly specialized form of cell division developed for the production of haploid gametes and essential for generating genetic diversity within species (27). In females, the progression of meiosis is not continuous; oocytes arrest in prophase I before ovulation, and arrest in metaphase II before fertilization. These arrests and the progression of meiosis are carefully controlled by pathways that are distinct from those that regulate mitosis. These processes are critical for germ cell 554 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0234474100
survival and normal ovarian folliculogenesis, as well as early embryonic development (28). Many lines of evidence have implicated cyclin B1 in directing the commitment to complete both metaphase I and metaphase II of meiosis as a regulatory component of the MPF complex. Cyclin B1 increases before each meiotic metaphase, and its ubiquitin-mediated degradation coincides with the metaphaseanaphase transition (18). Injection of cyclin B1 antisense mRNA causes defects during the completion of meiosis I and in the onset metaphase II (18). Similarly, meiosis I defects are evident in Mos knockout mice lacking a component of the cytostatic factor complex implicated in MPF stabilization, and in CDC25B phosphatase knockout mice lacking an activator of MPF (29, 30). Just as these experiments suggest that timely MPF activity is necessary for meiotic control, inappropriate cyclin B1 expression is sufficient to disrupt meiosis, as evidenced by sense mRNA
Fig. 7. Schematic representation of RFPL4 in complex with MPF and factors of the ubiquitin-mediated proteasomal degradation pathway. Interaction screens in yeast and CHO cell extract coimmunoprecipitation studies indicate that the C-terminal B30.2 domain of RFPL4 interacts with the E2 enzyme HR6A, the N-terminal D-box domain of cyclin B1, the 20S proteasome subunit 1, and ubiquitin (UbB). The E2 enzyme HR6A interacts with the N terminus of cyclin B1, which contains the cyclin box.
Suzumori et al.
microinjection experiments (18). Additionally, if proteasome activity is precluded by the addition of inhibitors (e.g., MG132 and lactacystin), cyclin B1 is not degraded and exit from metaphase I is blocked (31). Cyclin B1 degradation during meiosis in Saccharomyces cerevisiae, Aspergillus nidulans, Caenorhabitis elegans, and Xenopus laevis has been ascribed to the E3 ubiquitin ligase activity of APC. There are numerous factors that regulate this activity specifically in meiosis in these organisms. These include Ime2, Ama1p, and Mfr1 in yeast, and Emi1 in frogs (32–35). However, there are no reports to date that associate components or regulators of the APC specifically with meiosis in mammalian oocytes. Interestingly, certain mitotic APC substrates also function as regulators of APC activity. In yeast, the cell cycle regulators Cdc20p兾Fizzy and Cdc5p兾Polo-like kinase are targeted for ubiquitin-mediated proteolysis by the APC in late anaphase and G1, but first are required for ubiquitination of other APC substrates (reviewed in ref. 36). Along these lines, APC activity is regulated by Cdc2兾cyclin B phosphorylation in Xenopus egg extracts (37). In mice, it is thought that activation of mitotic APC depends on phosphorylation accomplished by polo-like kinase downstream of MPF (38). Our studies raise the possibility that RFPL4-mediated ubiquitination pathways are directly regulated by a cyclin B1-associated complex.
Thus, RFPL4 seems to represent an important mediator of a protein degradation pathway in mammalian oocytes, physically associating with the E2 enzyme HR6A, the target protein cyclin B1, and the proteasome subunit PMSB1 (Fig. 7). Other proteins that interact with RFPL4 as E3 target proteins or E3 complex regulators, the essential roles of RFPL4 and APC and their phosphorylation in mammalian oocytes and early embryonic development in vivo, and the relevance of RFPL4 homologs to gametogenesis or somatic cell cycle control remain to be discovered. The generation of Rfpl4 knockout mice will better define the roles of RFPL4 in oocyte protein degradation pathways, further the identification of additional RFPL4 target proteins, and clarify the role of RFPL4 in female fertility.
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We thank Karen Wigglesworth, Frank Pendola, and John Eppig for assistance recovering oocyte cDNA, Shlomi Lazar for critical reading of the manuscript, Michael Mancini and the Baylor College of Medicine Integrated Microscopy Core for assistance with deconvolution microscopic imaging, and Shirley Baker for assistance with manuscript preparation. This work was supported in part by National Institutes of Health grants (to M.M.M.) and the Specialized Cooperative Centers Program in Reproduction Research (HD07495). K.H.B. is a student in the Medical Scientist Training Program at Baylor College of Medicine and is supported in part by National Institutes of Health Training Grant GM07330. W.Y. is supported by a postdoctoral fellowship from the Ernst Schering Research Foundation.
