RFPL4 interacts with oocyte of the -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 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 . To study the expression and functions of RFPL4 protein, we (7–9). Mutations in the human ubiquitin-specific protease performed immunolocalizations and used yeast two-hybrid and 9 Y (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 detects RFPL4 in ovaries, oocytes, and pGBKT7 (MYC-tagged) and pGADT7 (hemagglutinin-tagged) two-cell embryos (Fig. 1) but fails to detect a band in other vectors were used as templates for in vitro transcription͞ samples. This finding is consistent with our Northern blot translation using [35S]Met and the TNT T7 Coupled Reticulo- analysis showing that Rfpl4 mRNA is ovary-specific (6). cyte Lysate System (Promega). In vitro translated proteins were combined at room temperature for 1 h, and reciprocal coimmu- RFPL4 Protein Is Expressed in Growing Oocytes and Early Embryos. noprecipitation experiments were performed by using mouse RFPL4 protein is located in growing oocytes in both wild-type Ϫ Ϫ anti-MYC monoclonal or rabbit anti-hemagglutinin polyclonal and Gdf9 / ovaries, beginning at the primary follicle stage and antibodies (CLONTECH). extending through the preovulatory follicle stage (Fig. 2 A–C). Deconvolution microscopy was used to assess RFPL4 protein Cell Culture, Plasmids, and Transfection. Chinese hamster ovary expression in oocytes throughout meiotic maturation and in (CHO)-K1 cells (American Type Culture Collection, Manassas, early preimplantation embryos. Fully grown oocytes were eval- BIOLOGY VA) were cultured in Dulbecco’s modified Eagle’s medium͞ uated before the resumption of meiosis (GV stage) (Fig. 2D) and DEVELOPMENTAL Ham’s F-12 (DMEM͞F-12) containing 10% FBS and grown to after ovulation and progression to metaphase II (Fig. 2G). In 90–95% confluence in 6-cm dishes. To express tagged proteins, GV stage oocytes, RFPL4 protein is located in the cytoplasm and mouse cDNAs were inserted into pCMV-Tag4A͞FLAG-C and nucleus, and seems relatively excluded from the nucleolus (Fig. pCMV-Tag5A͞MYC-C vectors (Stratagene) and transiently 2D); after nuclear membrane breakdown, RFPL4 is detected transfected by using LipofectAMINE 2000 (Invitrogen). Twenty- throughout the oocyte (Fig. 2G). No expression was discernable four hours after transfection, cells were harvested, processed in in oocyte-associated granulosa cells (data not shown). In addi- lysis buffer [50 mM Tris⅐HCl, pH 7.4͞150 mM NaCl͞1mM tion, the RFPL4 protein is degraded in two-cell stage and EDTA͞1% Triton X-100͞protease inhibitor mixture (Sigma)], eight-cell stage embryos (Fig. 2 E and F). Expression of RFPL4 and analyzed by immunoprecipitation and SDS͞PAGE. was quantified by comparing immunofluorescent signal intensi- ties in GV stage oocytes, metaphase II oocytes, and two-, four-, Cell Extract Immunoprecipitation and Western Blot Analysis. Immu- and eight-cell embryos (Fig. 3). These data strongly suggest a noprecipitations were performed by using the FLAG-Tagged specific role for RFPL4 in oogenesis and oocyte meiosis. Protein Immunoprecipitation kit (Sigma) as described by the manufacturer. The bound antibody was detected by the ECL Yeast Two-Hybrid Screening of Mouse cDNA Libraries. The full- detection kit (Amersham Pharmacia). length ORF of mouse Rfpl4 (Fig. 4A) corresponding to amino acid residues 1–287 was subcloned into the pGBKT7 vector for Results expression as a GAL4 DNA binding fusion protein. Ovarian and Production of RFPL4 Protein and Specificity of the Anti-RFPL4 Anti- oocyte cDNA libraries were subcloned into the pGADT7 vector body. We produced recombinant full-length mouse RFPL4 pro- to be expressed as transactivation domain fusion proteins. In this tein in Escherichia coli and isolated the product by using His tag yeast two-hybrid system, interactions between RFPL4 and pro- affinity. This reagent was used to generate polyclonal goat teins encoded by library cDNAs are expected to reconstitute antibodies. Affinity and specificity of the anti-RFPL4 antibody transactivating complexes, which bind to DNA and promote were tested by Western blot analysis. Total protein extracts transcription of selectable markers. To identify RFPL4- prepared from adult mouse wild-type heart, liver, spleen, testis, interacting proteins, we screened Ϸ1 ϫ 106 ovary cDNA trans-

Suzumori et al. PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 ͉ 551 Downloaded by guest on September 25, 2021 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)IntheGdf9 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 Ͼ600 colonies grew on Leu-͞ mant cDNAs corresponded to PSMB1. Other sequences iden- Trp-͞Ade-͞His-͞X-␣-Gal selection plates, and 27 of the isolated tified in these screens are provided in Table 1, which is published plasmids with inserts Ͼ500 bp were sequenced. Seven of these as supporting information on the PNAS web site, www.pnas.org. sequences were in-frame portions of the proteasome subunit ␤, type 1 (PSMB1) coding sequence (U60824). RFPL4-Interacting Proteins in Yeast. Based on the above protein– protein interactions and the primary structure of RFPL4 (Fig. We also screened Ϸ1 ϫ 106 oocyte cDNA transformants by using the pGBKT7-RFPL4 bait. A total of Ͼ200 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࿝011664). One of the transfor-

Fig. 3. Quantitative analysis of RFPL4 immunofluorescence in oocytes and Fig. 4. Truncation constructs of RFPL4 and cyclin B1. (A) Schematic repre- early embryos. High levels of RFPL4 are detected in GV stage oocytes (GV) sentation of RFPL4, and ⌬C79, ⌬N86, ⌬N79⌬C155, and ⌬N79 derivatives. The and metaphase II (MII) oocytes. RFPL4 signal is diminished in two-cell (2c) and RING finger-like region and B30.2 domain are shown as gray and black boxes, four-cell (4c) embryos, and is not detected in eight-cell (8c) morula. Average respectively. (B) Schematic representation of cyclin B1, and ⌬C198, and ⌬N251 intensities after 0.5-s exposures are given with prebleed intensities subtracted. derivatives. The destruction box (D-box) and cyclin box are shown as gray and Error bars represent the SEM of each sample (n ϭ 10). black boxes, respectively.

552 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0234474100 Suzumori et al. Downloaded by guest on September 25, 2021 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 C- terminal 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 signifi- cantly 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).

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 Fig. 5. The strength of protein–protein interactions assessed by cotransfor- polymerase from pGBKT7 and pGADT7 templates without mant and yeast mating fluorescence assays. After inoculation, we checked the initial fluorescence, allowed the cells to grow for 40 h, and then rechecked DNA binding or transactivation domains. As positive control fluorescence of an oxygen sensitive dye by using the MATCHMAKER Biosensor vectors, we used pGBKT7-murine p53 (MYC epitope-tagged) kit (CLONTECH). Fluorescence was excited at 485 nm, and emission was read and pGADT7-SV40 large T (hemagglutinin epitope-tagged). 35 at 630 nm. To find the fold increase in fluorescence, an indication of yeast [ S]methionine was included in translation mixtures to generate growth in selection media, we divided the fluorescence intensity at time t ϭ products detectable by autoradiography. ϭ ͞ 40 h by the initial intensity recorded at time t 0 h [Fluorescence(t40) RFPL4 (32 kDa) interacts with HR6A (17 kDa) and full- Fluorescence(t0)]. The values shown are the means of triplicate measurements length cyclin B1 (47 kDa) and HR6A binds full length cyclin B1 of three independent transformants. Standard deviations are indicated by the (Fig. 8, which is published as supporting information on the BIOLOGY error bars. The pGBKT7-murine p53 (p53) and pGADT7-SV40 large T-antigen PNAS web site). Interactions between UbB (33 kDa) and RFPL4 DEVELOPMENTAL (Large T) were used for positive control. (A) Cotransformant assay. (B and C) Yeast mating assay. (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 4A), we proposed that RFPL4 could act as an E3 ubiquitin ligase hybrid screen were not verifiable by cotransformant assays and to transfer ubiquitin from the HR6A E2 conjugating enzyme to coimmunoprecipitation approaches (Table 1). targets such as cyclin B1. To test interactions between RFPL4 and HR6A, cyclin B1, PSMB1, or UbB in yeast, we cotrans- Protein Interactions in CHO Cells. To confirm that RFPL4 binds to formed pGBKT7-RFPL4 and pGADT7 constructs and assessed HR6A, cyclin B1, and PSMB1 in mammalian cells, coimmuno- precipitation studies were performed by using extracts of tran- growth with Leu-͞Trp-͞Ade-͞His-͞X-␣-Gal selection. To assess siently transfected CHO cells. The interaction between HR6A the strength of each interaction, we used a fluorometric method (17 kDa) and cyclin B1 (47 kDa) was also studied by this for measuring yeast growth after cotransformation and mating. approach. Anti-FLAG antibodies could coimmunoprecipitate We considered that strong protein–protein interactions would FLAG-tagged, full-length RFPL4 bound to MYC-tagged result in a 3-fold increase in dye fluorescence over a 40-h HR6A, cyclin B1, or PSMB1 (25 kDa) from lysates of CHO cells incubation period. Cotransformants of pGBKT7-murine p53 cotransfected with these constructs (Fig. 6A). Likewise, the and pGADT7-SV40 large T antigen were used as a positive anti-FLAG antibodies could coimmunoprecipitate FLAG- Ͼ control ( 5-fold increase), and cotransformants of pGBKT7- tagged cyclin B1 and MYC-tagged HR6A or FLAG-tagged RFPL4 and empty pGADT7 vector were used as a negative cyclin B1 and MYC-tagged RFPL4 (Fig. 6B). No deleterious Ϸ control ( 1-fold). We found rapid growth of cotransformants effects were observed in any transiently transfected cells after with pGBKT7-RFPL4 and pGADT7-HR6A, -N-terminal cy- 48 h of expression. Because the RFPL4 B30.2 domain interacts clin B1 (CCNB1⌬C198; Fig. 4B), -PSMB1, or -UbB, but not with cyclin B1 and HR6A in vitro, we expressed a truncated form the cyclin B1 C terminus (CCNB1⌬N251) (Fig. 5A and data of RFPL4 that lacks the RING finger-like motif (RFPL4⌬N79), not shown). In contrast, yeast growth assays suggest along with cyclin B1 or HR6A; however, binding was not that HR6A interacts strongly with the C terminus of cyclin demonstrated in CHO cells (data not shown). In addition, we

Suzumori et al. PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 ͉ 553 Downloaded by guest on September 25, 2021 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 immuno- precipitated 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 survival and normal ovarian folliculogenesis, as well as early RFPL4 in CHO cells, but could not detect an interaction. These embryonic development (28). latter findings imply that these interactions are weak in CHO Many lines of evidence have implicated cyclin B1 in directing cells, possibly because of the presence of other factors that the commitment to complete both metaphase I and metaphase regulate the binding of these proteins. II of meiosis as a regulatory component of the MPF complex. Cyclin B1 increases before each meiotic metaphase, and its Discussion ubiquitin-mediated degradation coincides with the metaphase- Our laboratory recently reported the discovery of the Rfpl4 gene anaphase transition (18). Injection of cyclin B1 antisense mRNA (6), which we now demonstrate encodes an oocyte-specific causes defects during the completion of meiosis I and in the onset RING finger-like protein. RFPL4 protein accumulates to high metaphase II (18). Similarly, meiosis I defects are evident in Mos levels during oogenesis and is rapidly lost in early cleavage stage knockout mice lacking a component of the cytostatic factor embryos. Based on the primary amino acid sequence, we spec- complex implicated in MPF stabilization, and in CDC25B phos- ulated that RFPL4 may function as an E3 ubiquitin ligase to phatase knockout mice lacking an activator of MPF (29, 30). Just target proteins for proteasomal degradation. A yeast two-hybrid as these experiments suggest that timely MPF activity is neces- screen and immunoprecipitation studies conducted with in vitro sary for meiotic control, inappropriate cyclin B1 expression is translated protein and transiently transfected CHO cell protein sufficient to disrupt meiosis, as evidenced by sense mRNA 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 em- bryos 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 gener- ating genetic diversity within species (27). In females, the Fig. 7. Schematic representation of RFPL4 in complex with MPF and factors progression of meiosis is not continuous; oocytes arrest in of the ubiquitin-mediated proteasomal degradation pathway. Interaction screens in yeast and CHO cell extract coimmunoprecipitation studies indicate prophase I before ovulation, and arrest in metaphase II before that the C-terminal B30.2 domain of RFPL4 interacts with the E2 enzyme HR6A, fertilization. These arrests and the progression of meiosis are the N-terminal D-box domain of cyclin B1, the 20S proteasome subunit ␤1, and carefully controlled by pathways that are distinct from those that ubiquitin (UbB). The E2 enzyme HR6A interacts with the N terminus of cyclin regulate mitosis. These processes are critical for germ cell B1, which contains the cyclin box.

554 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0234474100 Suzumori et al. Downloaded by guest on September 25, 2021 microinjection experiments (18). Additionally, if proteasome Thus, RFPL4 seems to represent an important mediator of a activity is precluded by the addition of inhibitors (e.g., MG132 protein degradation pathway in mammalian oocytes, physically and lactacystin), cyclin B1 is not degraded and exit from associating with the E2 enzyme HR6A, the target protein cyclin metaphase I is blocked (31). B1, and the proteasome subunit PMSB1 (Fig. 7). Other proteins Cyclin B1 degradation during meiosis in Saccharomyces cer- that interact with RFPL4 as E3 target proteins or E3 complex evisiae, Aspergillus nidulans, Caenorhabitis elegans, and Xenopus regulators, the essential roles of RFPL4 and APC and their laevis has been ascribed to the E3 ubiquitin ligase activity of phosphorylation in mammalian oocytes and early embryonic APC. There are numerous factors that regulate this activity development in vivo, and the relevance of RFPL4 homologs to specifically in meiosis in these organisms. These include Ime2, gametogenesis or somatic cell cycle control remain to be dis- Ama1p, and Mfr1 in yeast, and Emi1 in frogs (32–35). However, covered. The generation of Rfpl4 knockout mice will better there are no reports to date that associate components or define the roles of RFPL4 in oocyte protein degradation path- regulators of the APC specifically with meiosis in mammalian ways, further the identification of additional RFPL4 target oocytes. Interestingly, certain mitotic APC substrates also func- proteins, and clarify the role of RFPL4 in female fertility. tion as regulators of APC activity. In yeast, the cell cycle ͞ ͞ We thank Karen Wigglesworth, Frank Pendola, and John Eppig for regulators Cdc20p Fizzy and Cdc5p Polo-like kinase are tar- assistance recovering oocyte cDNA, Shlomi Lazar for critical reading of geted for ubiquitin-mediated proteolysis by the APC in late the manuscript, Michael Mancini and the Baylor College of Medicine anaphase and G1, but first are required for ubiquitination of Integrated Microscopy Core for assistance with deconvolution micro- other APC substrates (reviewed in ref. 36). Along these lines, scopic imaging, and Shirley Baker for assistance with manuscript prep- APC activity is regulated by Cdc2͞cyclin B phosphorylation in aration. This work was supported in part by National Institutes of Health Xenopus egg extracts (37). In mice, it is thought that activation grants (to M.M.M.) and the Specialized Cooperative Centers Program in Reproduction Research (HD07495). K.H.B. is a student in the of mitotic APC depends on phosphorylation accomplished by Medical Scientist Training Program at Baylor College of Medicine and polo-like kinase downstream of MPF (38). Our studies raise the is supported in part by National Institutes of Health Training Grant possibility that RFPL4-mediated ubiquitination pathways are GM07330. W.Y. is supported by a postdoctoral fellowship from the Ernst directly regulated by a cyclin B1-associated complex. Schering Research Foundation.

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