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1 Tdrd3 regulates the progression of meiosis II through translational 2 control of Emi2 mRNA in mouse oocytes 3 4 Natsumi Takei1, Keisuke Sato1, Yuki Takada1, Rajan Iyyappan2, Andrej Susor2, Takehiro 5 Yamamoto3, and Tomoya Kotani1,4,* 6 7 1Biosystems Science Course, Graduate School of Life Science, Hokkaido University, 8 Sapporo 060-0810, Japan 9 2Laboratory of Biochemistry and Molecular Biology of Germ Cells, Institute of Animal 10 Physiology and Genetics, CAS, Rumburska 89, 277 21 Libechov, Czech Republic. 11 3Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, 12 Japan 13 4Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 14 060-0810, Japan 15 16 17 *Corresponding author. 18 Tomoya Kotani, 19 Department of Biological Sciences, Faculty of Science, Hokkaido University, 20 North 10 West 8, Sapporo, Hokkaido 060-0810, Japan 21 Tel.: +81-11-706-4455 22 Fax.: +81-11-706-4455 23 E-mail: [email protected] 24 25 26 Running title: Tdrd3 regulates oocyte meiosis 27 Key words: oocyte, meiosis, Emi2, cyclin B1, RNA granule, translational control 28
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29 ABSTRACT 30 After completion of meiosis I, the oocyte immediately enters meiosis II and forms a 31 metaphase II (MII) spindle without an interphase, which is fundamental for generating a 32 haploid gamete. Here, we identify tudor domain-containing protein 3 (Tdrd3) as a novel 33 regulator of oocyte meiosis. Although early mitotic inhibitor 2 (Emi2) protein has been 34 shown to ensure the meiosis I to II transition and the subsequent MII spindle formation 35 by inhibiting the anaphase-promoting complex/cyclosome (APC/C), how it accumulates 36 after meiosis I has remained unresolved. We isolated Tdrd3 as a protein directly binding 37 to Emi2 mRNA. In GV-stage mouse oocytes, Emi2 mRNA assembled into RNA 38 granules containing Tdrd3, while cyclin B1 mRNA, which was translated in early 39 meiosis I, formed different granules. Knockdown of Tdrd3 attenuated Emi2 synthesis in 40 meiosis II without affecting cyclin B1 synthesis in meiosis I. Moreover, Tdrd3-deficient 41 oocytes entered interphase and failed to form an MII spindle after completion of meiosis 42 I. Taken together, our results indicate the importance of Tdrd3-mediated translational 43 control of Emi2 mRNA, which promotes Emi2 synthesis in meiosis II, for the 44 progression of meiosis. 45
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46 INTRODUCTION 47 Gametes are generated through the two successive meiotic cell divisions, meiosis I and 48 meiosis II. In oocytes, meiosis is arrested at the prophase of meiosis I, and more than 49 ten thousand mRNAs are accumulated for oocyte growth and for the progression of 50 meiosis and embryonic development (Conti and Franciosi, 2018; Masui and Clarke, 51 1979; Meneau et al., 2020; Winata and Korzh, 2018). Fully grown germinal vesicle 52 (GV)-stage oocytes resume meiosis in response to hormonal stimulation. Shortly after 53 the resumption of meiosis, oocytes activate maturation/M-phase-promoting factor 54 (MPF), which consists of the catalytic subunit Cdc2 and regulatory subunit cyclin B 55 (Nurse, 1990). The kinase activity of MPF promotes chromosome condensation, 56 dissolution of the nuclear membrane, and formation of a metaphase I (MI) spindle 57 (Abrieu et al., 2001; Kotani and Yamashita, 2002; Ledan et al., 2001; Polanski et al., 58 1998). Degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) 59 partially inactivates MPF, resulting in the separation of homologous chromosomes. 60 Subsequently, the MPF activity is rapidly increased, and oocytes enter meiosis II and 61 form a metaphase II (MII) spindle without an interphase. In vertebrates, meiosis is 62 arrested again at MII by an activity called cytostatic factor (CSF) while awaiting 63 fertilization (Masui and Markert, 1971; Sagata, 1996). CSF stabilizes MPF activity 64 indirectly by inhibition of APC/C, preventing sister chromatid separation at MII 65 (Madgwick et al., 2004; Nixon et al., 2002). 66 Early mitotic inhibitor 2 (Emi2) has been identified as one of the components of CSF 67 in Xenopus and mouse oocytes (Madgwick et al., 2006; Schmidt et al., 2005; Shoji et 68 al., 2006; Tung et al., 2005). Emi2 was shown to inhibit APC/C by binding directly to 69 APC/C, resulting in the arrest of meiosis at MII in Xenopus (Ohe et al., 2010). Emi2 70 protein is absent in GV-stage immature Xenopus oocytes and accumulates in meiosis II 71 (Liu et al., 2006; Ohe et al., 2007). Previous studies showed that inhibition of Emi2 72 synthesis by injection of Emi2 small interfering RNA (siRNA) or antisense morpholino 73 oligo nucleotide (MO) resulted in failure in arrest at MII, while ectopic expression of 74 Emi2 in meiosis I resulted in the arrest of meiosis at MI in Xenopus and mouse oocytes 75 (Madgwick et al., 2006; Ohe et al., 2007; Shoji et al., 2006; Suzuki et al., 2010). These 76 studies indicate that the timing of Emi2 synthesis as well as Emi2 synthesis itself is 77 critical for APC/C inhibition in meiosis II. However, how and when Emi2 is synthesized 78 from the mRNA stored in oocytes remain unresolved.
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79 Of the mRNAs that have accumulated in GV-stage oocytes, thousands of mRNAs 80 have been shown to be translationally repressed after transcription (Chen et al., 2011). 81 After the resumption of meiosis, hundreds of mRNAs begin to be translated in meiosis I 82 and more than one thousand mRNAs begin to be translated in meiosis II (Chen et al., 83 2011; Luong et al., 2020). cyclin B1 mRNA is one of the mRNAs translated in early 84 meiosis I (Haccard and Jessus, 2006; Han et al., 2017; Kotani et al., 2013; Nakahata et 85 al., 2003; Yang et al., 2017). The 3’ untranslated region (3’UTR) of many dormant 86 mRNAs including cyclin B1 mRNA contains the cytoplasmic polyadenylation element 87 (CPE), which is recognized by CPE-binding proteins (CPEBs) (Richter, 2007). CPEB1 88 has been shown to mediate the cytoplasmic polyadenylation of dormant mRNAs with 89 short poly(A) tails after resumption of meiosis, resulting in translational activation of 90 target mRNAs (Gebauer et al., 1994; Hake and Richter, 1994; Stebbins-Boaz et al., 91 1996; Tay et al., 2000). Although a large number of dormant mRNAs contain CPEs in 92 their 3’UTRs, they are translated in different periods, suggesting that other mechanisms 93 in addition to the mechanism that depends on the CPEB1 regulate the timing of 94 translational activation of distinct mRNAs. 95 Recent studies have shwon that cytoplasmic RNA granules such as stress granules 96 and processing bodies (P-bodies) mediate post-transcriptional regulations including 97 stabilization and degradation of assembled mRNAs (Anderson and Kedersha, 2009; 98 Buchan and Parker, 2009). We previously showed that the translation of cyclin B1 99 mRNA in meiosis I was regulated by the formation and disassembly of RNA granules 100 (Kotani et al., 2017; Kotani et al., 2013). Mad2 mRNA was shown to be translated in a 101 similar period, and both mRNAs were targets of an RNA-binding protein, Pumilio1 102 (Pum1) (Takei et al., 2020). Pum1 was shown to form aggregates in GV-stage oocytes 103 and regulate the translation of cyclin B1 and Mad2 mRNAs through assembly and 104 dissolution of the aggregates. 105 The 3’UTR of Xenopus emi2 mRNAs contains several CPEs that were shown to bind 106 to CPEB1 (Tung et al., 2007). Although CPEB1 was shown to direct the 107 polyadenylation of many dormant mRNAs after resumption of meiosis, this protein is 108 degraded in meiosis I in Xenopus and mouse oocytes (Chen et al., 2011; Mendez et al., 109 2002; Setoyama et al., 2007). In Xenopus oocytes, polyadenylation of dormant mRNAs 110 in meiosis II was shown to be directed by CPEB4, which accumulated in the later stage 111 of meiosis I (Igea and Mendez, 2010). However, injection of antisense Cpeb4 MO into
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112 GV-stage oocytes had no significant effect on the progression of meiosis in mouse 113 oocytes (Chen et al., 2011), suggesting that CPEB4 is not mainly involved in the Emi2 114 mRNA translation in mouse oocytes. 115 In this study, we identified tudor domain-containing protein 3 (Tdrd3) as a novel 116 protein that binds to Emi2 mRNA in mouse oocytes. Emi2 mRNA was found to 117 assemble into granules, which were different from those of cyclin B1 mRNA. Although 118 HuR and HuB, components of stress granules (Gallouzi et al., 2000; Markmiller et al., 119 2018), and CPEB1 interacted with both Emi2 and cyclin B1 mRNAs, Tdrd3 bound to 120 and colocalized with Emi2, but not cyclin B1, mRNA. Knockdown of Tdrd3 attenuated 121 Emi2 synthesis in meiosis II but did not affect the synthesis of cyclin B1 and Mad2 in 122 meiosis I. Consistent with this, Tdrd3-deficient oocytes entered interphase and failed to 123 form an MII spindle after completion of meiosis I. Taken together, our results indicate 124 that the assembly of Tdrd3-containing granules controls the translation of Emi2 mRNA 125 in meiosis II, through which oocytes promote the progression of meiosis II. 126 127 RESULTS 128 Temporally controlled translation of Emi2 mRNA is prerequisite for the generation 129 of a fertilizable gamete 130 To assess how the function of Emi2 is temporally regulated in meiosis, we first analyzed 131 the accumulation of Emi2 protein in mouse oocytes by raising antibodies against the N- 132 terminal region of mouse Emi2 (amino acids 1-366). Affinity-purified anti-Emi2 133 antibodies recognized a protein with an apparent molecular mass of ~90 kDa in 134 immunoblots of MII- stage oocytes but not GV-stage oocytes (Fig. S1A), which 135 corresponds to the size of Emi2 shown in a previous study (Madgwick et al., 2006). The 136 intensity of this signal was significantly reduced when the antibody was preincubated 137 with recombinant Emi2 (Fig. S1B) and when the translation of Emi2 mRNA was 138 prevented by the antisense MO (Fig. 1D), confirming that the antibodies specifically 139 recognize Emi2. Emi2 was not detected at 0 h (GV stage) and 8 h (MI stage) after 140 resumption of meiosis, while a large amount of Emi2 was detected at 18 h (MII stage) 141 (Fig. 1A). In contrast, the amount of cyclin B1 was increased at the MI stage and 142 peaked at the MII stage (Fig. 1A), consistent with the results in previous studies 143 (Ganesh et al., 2020; Takei et al., 2020; Yang et al., 2017). 144 A poly(A) test (PAT) assay showed that Emi2 mRNA was polyadenylated only
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145 slightly at 4 h (prometaphase I, PMI, stage) and was significantly polyadenylated at 18 146 h (MII stage) (Fig. 1B). In contrast, cyclin B1 mRNA was rapidly polyadenylated at the 147 PMI stage and polyadenylation peaked at the MII stage (Fig. 1B). Polysomal 148 fractionation analysis showed that the amount of Emi2 mRNA in the polysomal fraction 149 increased by approximately 5 fold from the GV stage to the MII stage (Fig. 1C), 150 although monosomal and polysomal occupation of Emi2 mRNA was equal between the 151 GV and MII stages (Fig. S1C). Taken together, these results indicate that Emi2 mRNA 152 is translationally repressed in meiosis I with short poly(A) tails and is translated in 153 meiosis II possibly through the polyadenylation of mRNA. 154 A recent study demonstrated that antisense MOs targeting the 3’-end sequences of 155 mRNAs prevent the polyadenylation of mRNAs (Wada et al., 2012). To assess the 156 importance of polyadenylation of Emi2 mRNA, we injected an antisense Emi2 MO that 157 targets the terminal sequence of the Emi2 mRNA 3’UTR (Emi2-3’UTR-MO) and an 158 Emi2 MO that contains 5-nts mismatches (Emi2-5mm-MO) as a control (Fig. 1D) into 159 GV-stage oocytes. Immunoblot analysis showed that the synthesis of Emi2 was 160 effectively inhibited by Emi2-3’UTR-MO, but not by Emi2-5mm-MO, at 28 h after 161 resumption of miosis (Fig. 1D). Immunostaining showed that the control oocytes had 162 extruded the first polar body and had formed an MII spindle with chromosomes aligned 163 on the equatorial plate at 28 h after resumption of meiosis (Fig. 1E), indicating that the 164 oocytes are arrested at MII. In contrast, the oocytes injected with Emi2-3’UTR-MO 165 possessed a pronucleus-like structure with the first polar body (Fig. 1E). At 48 h after 166 resumption of meiosis, the Emi2-3’UTR-MO-injected oocytes entered into the two-cell 167 stage. These phenotypes were consistent with the defects observed in oocytes in which 168 Emi2 was knocked down by Emi2-siRNA or Emi2-MO targeting the translational start 169 site of Emi2 mRNA (Madgwick et al., 2006; Shoji et al., 2006). These results indicate 170 that Emi2 is translated in meiosis II in a polyadenylation-dependent manner, which is 171 required for the entry into meiosis II and the prevention of parthenogenetic 172 development. 173 We then confirmed the effect of precocious Emi2 synthesis in meiosis I by injecting 174 mRNA encoding GFP or GFP-Emi2 into GV-stage oocytes. In oocytes expressing GFP, 175 81.1% of the oocytes underwent normal meiotic progression and were arrested at MII 176 (Fig. 1F and G). In contrast, 83.5% of the oocytes expressing GFP-Emi2 were arrested 177 at MI (Fig. 1F and G). In these oocytes, GFP-Emi2 was localized at the MI spindle (Fig.
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178 1F). These results are consistent with results of previous studies (Madgwick et al., 2006; 179 Suzuki et al., 2010). Taken together, our results indicate that the temporally controlled 180 translation of Emi2 mRNA defines the timing and duration of Emi2 function, which is 181 prerequisite for meiosis I to II transition, progression of meiosis II and arrest at MII. 182 183 Emi2 mRNA forms cytoplasmic granules that are distinct from cyclin B1 RNA 184 granules 185 To elucidate the mechanisms by which the translation of Emi2 mRNA is temporally 186 regulated, we first analyzed the distribution of Emi2 mRNA in mouse oocytes. Previous 187 studies demonstrated that dormant cyclin B1 and Mad2 mRNAs assemble into granules 188 in GV-stage oocytes, and the disassembly of these granules after resumption of meiosis 189 regulates the timing of translational activation of assembled mRNAs (Kotani et al., 190 2013; Takei et al., 2020). Emi2 mRNA was detected in GV-stage oocytes by the highly 191 sensitive in situ hybridization with the tyramide signal amplification (TSA) system (Fig. 192 2A). Fluorescent in situ hybridization (FISH) with the TSA system showed that Emi2 193 mRNA formed RNA granules in the oocyte cytoplasm (Fig. 2B). Interestingly, double 194 FISH analysis showed that Emi2 and cyclin B1 mRNAs formed different granules (Fig. 195 2C). 196 After the resumption of meiosis, large parts of cyclin B1 RNA granules rapidly 197 disassembled at PMI as observed in previous studies (Kotani et al., 2013; Takei et al., 198 2020), while the number of Emi2 RNA granules was maintained at this stage (Fig. 2D 199 and E). In the MII stage, Emi2 RNA granules had almost completely disappeared as in 200 the case of cyclin B1 (Fig. 2D and E). Since the total amount of Emi2 mRNA was not 201 decreased in MII-stage oocytes (Fig. S1C), the disappearance of Emi2 RNA granules 202 indicates granule disassembly. The disassembly of Emi2 RNA granules coincided with 203 the polyadenylation of Emi2 mRNA (Fig. 1B). These results suggest that translation of 204 Emi2 mRNA is regulated through formation and disassembly of RNA granules as in the 205 case of cyclin B1 and Mad2 mRNA (Kotani et al., 2013; Takei et al., 2020). However, 206 Emi2 RNA granules disassembled in later stages compared to the rapid disassembly of 207 cyclin B1 RNA granules, suggesting differences in the assembly and regulation of these 208 granules. 209 210
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211 Pum1 binds to cyclin B1 mRNA, but not to Emi2 mRNA, while HuR and HuB bind 212 to both mRNAs 213 To address the difference and similarity of Emi2 and cyclin B1 RNA granules, we 214 analyzed RNA-binding proteins interacting with Emi2 and/or cyclin B1 mRNAs. The 215 previous study demonstrated that Pum1 interacts with cyclin B1 and Mad2 mRNAs and 216 regulates the translational activation of these mRNAs in early meiosis I (Takei et al., 217 2020). To examine whether Pum1 interacts with Emi2 mRNA, we performed an 218 immunoprecipitation assay followed by RT-PCR (IP/RT-PCR). As expected, cyclin B1 219 mRNA was detected in precipitation with an anti-Pum1 antibody, while neither Emi2 220 mRNA nor α-tubulin mRNA was detected in the precipitation (Fig. 3A), indicating that 221 Emi2 mRNA is not a target of Pum1. Recent studies have demonstrated that different 222 types of cytoplasmic granules share several components (see a review Buchan and 223 Parker, 2009). We then analyzed the interaction of HuR and HuB proteins with Emi2 224 and cyclin B1 mRNAs, since HuR and HuB are components of stress granules and are 225 expressed in the ovary (Colombrita et al., 2013; Hinman and Lou, 2008) and HuR was 226 shown to be colocalized with cyclin B1 RNA granules in zebrafish oocytes (Kotani et 227 al., 2013). Emi2 and cyclin B1 mRNAs, but not α-tubulin mRNA, were detected in 228 precipitations with an anti-HuR or anti-HuB antibody (Fig. 3B and C), indicating that 229 HuR and HuB bind to both Emi2 and cyclin B1 mRNAs. These results suggest that HuR 230 and HuB are components common to Emi2 and cyclin B1 RNA granules, whereas Pum1 231 is specific to cyclin B1 RNA granules. 232 233 Isolation of Tdrd3 as a protein directly binding to Emi2, but not cyclin B1, mRNA 234 The results showing that Pum1 binds to cyclin B1, but not Emi2, mRNA suggested that 235 the precise regulation of distinct RNA granules is achieved by components specific to 236 each RNA granule. To isolate proteins specific to Emi2 RNA granules, we performed an 237 RNA pull-down assay by using in vitro-synthesized RNAs of Emi2 and cyclin B1 238 3’UTRs with oocyte extracts. In this assay, we used Xenopus oocytes since sufficient 239 materials were not obtained by using mouse oocytes. Mass spectrometry analysis 240 identified 71 candidate proteins specifically binding to Emi2 mRNA, 162 candidate 241 proteins binding to both mRNAs, and 65 candidate proteins specifically binding to 242 cyclin B1 mRNA (Fig. 3D and Table S1). Gene ontology (GO) terms of the proteins 243 were associated with RNA metabolism and translational control (Fig. 3E). Moreover,
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244 HuB and CPEB1 were isolated as proteins binding to both mRNAs, being consistent 245 with the results of in vivo analysis (Fig. 3C) and of in vitro UV cross-linking assay (see 246 Fig. 4C) and evaluating the results of RNA pull-down assay. 247 Tudor domain-containing protein 3 (Tdrd3), a modular protein identified on the basis 248 of its tudor domain of approximately 60 amino acids (Fig. 4A) (Goulet et al., 2008; 249 Linder et al., 2008), was isolated as one of the candidates specifically binding to Emi2 250 mRNA. Tdrd3 possesses putative nucleic acid recognition motifs (DUF and OB-fold 251 motifs), a ubiquitin-binding associated (UBA) domain, a fragile X mental retardation 252 protein (FMRP)-interacting domain, and an exon junction complex binding motif 253 (EBM) (Fig. 4A). We focused on Tdrd3 since the expression of Tdrd3 mRNA and that 254 of Tdrd3 protein were confirmed in GV-stage mouse oocytes (Fig. 4B) and Tdrd3 has 255 been shown to assemble into stress granules (Goulet et al., 2008; Linder et al., 2008), 256 although its physiological function has remained unknown. Immunoblot analysis 257 showed that the expression levels of Tdrd3 were not changed in GV- and MII-stage 258 oocytes (Fig. 4B). 259 We first examined whether Tdrd3 directly binds to Emi2 mRNA by performing an in 260 vitro UV cross-linking assay, since a direct interaction between Tdrd3 and RNA has not 261 been tested. RNA probes consisting of the 3’UTR of Emi2 and cyclin B1 were 262 incubated with Flag-tagged Tdrd3 and CPEB1. Both RNA probes were associated with 263 CPEB1 (Fig. 4C), consistent with results of previous studies in Xenopus oocytes 264 (Stebbins-Boaz et al., 1996; Tung et al., 2007). Since a large fraction of full-length 265 Tdrd3 was destructed in this assay (Fig. S2A), we prepared deleted forms of Tdrd3 that 266 consisted of N-terminal (1-473 aa) and C-terminal (246-743 aa) regions of Tdrd3 267 (Tdrd3-N and -C) (Fig. 4A). Immunoblot analysis showed that Tdrd3-C was divided 268 into many fragments when it was translated in a rabbit reticulocyte lysate system 269 regardless of the position of the Flag-tag, while Tdrd3-N was not destructed (Fig. S2B). 270 Therefore, we used Flag-tagged Tdrd3-N for the UV cross-linking assay. The Emi2 271 RNA probe was associated with Tdrd3-N-Flag, whereas the cyclin B1 RNA probe was 272 not (Fig. 4D), indicating that Tdrd3 specifically and directly interacts with Emi2 273 mRNA. 274 275 Tdrd3 colocalized with Emi2, but not cyclin B1, RNA granules 276 To examine the interaction between Tdrd3 and Emi2 mRNA in vivo, we first performed
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277 IP/RT-PCR by using anti-Tdrd3 antibodies. Unfortunately, Tdrd3 was not precipitated 278 with antibodies raised against three different antigens. We then analyzed the distribution 279 of Tdrd3 in the oocyte cytoplasm by immunostaining of mouse ovaries. Super- 280 resolution microscopy (structured illumination microscopy, SIM) showed that Tdrd3 281 was distributed in the cytoplasm of GV-stage oocytes as many particles (Fig. 5A). Since 282 these Tdrd3 particles seemed to form clusters in the cytoplasm, we calculated the 283 average distance of five neighboring particles and compared it with that of randomly 284 distributed dots (Fig. S3). The distance of Tdrd3 particles was significantly shorter than 285 that of randomly distributed dots (Fig. 5C), suggesting the formation of Tdrd3 clusters. 286 Simultaneous detections of Tdrd3 with Pum1 showed that particles of Tdrd3 were not 287 overlapped with Pum1 aggregates (Fig. 5B). The size of Tdrd3 particles appeared to be 288 smaller than that of Pum1 (Fig. 5D), and the shape of Tdrd3 particles tended to be round 289 compared to that of Pum1 (Fig. 5E), which was shown to assemble into solid-like 290 highly structured aggregates (Takei et al., 2020) (see also Fig. 5G). FISH analysis 291 showed that Tdrd3 particles colocalized with Emi2 RNA granules (Fig. 5F, arrows), but 292 no overlap was observed with cyclin B1 RNA granules (Fig. 5F). In contrast, Pum1 293 aggregates surrounded and overlapped with cyclin B1 RNA granules (Fig. 5G, arrows), 294 being consistent with the results of the previous study (Takei et al., 2020), while no 295 overlap was observed with Emi2 RNA granules (Fig. 5G), supporting the results of 296 IP/RT-PCR analysis (Fig. 3A). These results demonstrate that Tdrd3 is a component 297 specific to Emi2 RNA granules. 298 299 Tdrd3 controls the Emi2 mRNA translation in meiosis II 300 To assess whether Tdrd3 plays a role in the regulation of Emi2 mRNA, we injected an 301 anti-Tdrd3 antibody or control IgG into GV-stage oocytes and induced the resumption 302 of meiosis. Immunoblot analyses showed that the initial accumulation of Emi2 at 12 h 303 after resumption of meiosis was prevented by the anti-Tdrd3 antibody (Fig. 6A and B), 304 although the amount of Emi2 was recovered at 18 h. These results suggest that Tdrd3 is 305 involved in the translational activation of Emi2 mRNA in meiosis II. However, the 306 oocytes injected with the anti-Tdrd3 antibody extruded a polar body in the normal time 307 course and seemed to be arrested at MII as in the case of oocytes injected with IgG (Fig. 308 S4A and B). We speculated that this antibody only partially interfered with the Tdrd3 309 function and that the recovery of Emi2 accumulation uncovered the actual function of
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310 Tdrd3 in the progression of meiosis. To overcome this, we first attempted to knock 311 down Tdrd3 by antisense MOs, but injection of the antisense Tdrd3 MO did not reduce 312 the amount of Tdrd3 even in oocytes incubated for 18 h after injection (Fig. S4C), 313 suggesting that Tdrd3 is stable in oocytes. We then attempted to knock down Tdrd3 by 314 using the Trim-Away protein degradation system (Clift et al., 2017) with the anti-Tdrd3 315 antibody. This system allows rapid depletion of an endogenous protein by TRIM- 316 mediated degradation of the antibody-target protein complex. 317 Injection of mRNA encoding a modified version of TRIM21, Piranha, with the anti- 318 Tdrd3 antibody effectively reduced the amount of Tdrd3, while no reduction was 319 observed with injection of Piranha mRNA with control IgG (Fig. 6C). These oocytes 320 formed a pronucleus-like structure and a monopolar spindle after extruding the polar 321 body, while no defect was observed in control oocytes injected with Piranha mRNA and 322 IgG (Fig. 6D and E). The accumulation of Emi2 was attenuated throughout meiosis II 323 by the knockdown of Tdrd3 (Fig. 6F and G). In contrast, the amounts of cyclin B1 and 324 Mad2 in meiosis I were not affected (Fig. 6F and G). The amount of cyclin B1, but not 325 that of Mad2, was reduced in meiosis II, which would be caused by the activation of 326 APC/C in meiosis II due to the reduced levels of Emi2. The defect in meiosis II was 327 almost completely recovered by GFP-Emi2 expressed after completion of meiosis I 328 (Fig. 6D and E). Taken together, these results indicate that Tdrd3 is required for the 329 translation of Emi2 mRNA in meiosis II and regulates the progression of meiosis II 330 thorough the synthesis of Emi2 protein after completion of meiosis I until MII arrest. 331 332 DISCUSSION 333 In Xenopus oocytes, Emi2 protein has been shown to accumulate after meiosis I and 334 prevent APC/C during the progression of meiosis II (Liu et al., 2006; Ohe et al., 2007; 335 Tung et al., 2007). Analyses using reporter mRNAs suggested that the translational 336 control of Emi2 mRNA, which would be dependent on CPE and CPEB1, was important 337 for the timing of Emi2 accumulation (Ohe et al., 2007; Tung et al., 2007). However, 338 whether the translation of endogenous Emi2 mRNA is involved in the timely controlled 339 protein accumulation has been obscure. Moreover, whether Emi2 mRNA is regulated at 340 the translational level in mouse oocytes has remained completely unknown. In this 341 study, we demonstrated that Emi2 mRNA is translationally repressed with short poly(A) 342 tails in meiosis I and translationally activated in a polyadenylation-dependent manner in
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343 meiosis II in mouse oocytes (Fig. 1). Tdrd3 was identified as a novel protein binding 344 specifically to Emi2 mRNA and was shown to colocalize with Emi2 RNA granules in 345 GV-stage oocytes (Figs. 2, 3, 4 and 5). Knockdown of Tdrd3 caused failure in meiosis II 346 entry and MII spindle formation through attenuation of Emi2 synthesis (Fig. 6). 347 Collectively, our results provide evidence that the Emi2 accumulation in meiosis II is 348 actually regulated by the translational control of Emi2 mRNA that is deposited in 349 oocytes. Moreover, Tdrd3-mediated translation of Emi2 mRNA is fundamental for the 350 generation of a fertilizable gamete. 351 FISH analyses of mouse ovaries showed that Emi2 mRNA assembled into RNA 352 granules, which are different from those of cyclin B1, in GV-stage oocytes (Fig. 2). 353 IP/RT-PCR and the RNA pull-down assay followed by mass spectrometry analysis 354 suggested that Emi2 and cyclin B1 RNA granules share certain proteins, but each 355 granule also possesses specific components (Figs. 3 and 4). These results suggest that 356 accurate translational control of assembled mRNAs is achieved by the assembly of 357 common and specific components of RNA granules. In contrast to the specific 358 components such as Pum1 and Tdrd3, HuR and HuB were shown to bind to both cyclin 359 B1 and Emi2 mRNAs (Fig. 3B and C). HuR is widely expressed in various cells and 360 tissues and has been shown to be a component of stress granules (Gallouzi et al., 2000; 361 Papadopoulou et al., 2013). In HuR-knockdown cultured human cells, the size of stress 362 granules generally became much smaller, and the number of granules decreased, 363 indicating that HuR is a key component in assembling stress granules (Fujimura et al., 364 2009). HuB is expressed specifically in neurons and in the testis and ovary. Recent 365 studies have shown that HuB is a component of neuronal stress granules (Markmiller et 366 al., 2018) and functions in translational repression of many mRNAs in mouse oocytes 367 (Chalupnikova et al., 2014). Therefore, HuR and HuB may function in assembling and 368 translationally repressing cyclin B1 and Emi2 mRNAs in mouse oocytes. Our UV cross- 369 linking assay showed that CPEB1 also binds to both Emi2 and cyclin B1 mRNAs (Fig. 370 4C). In cultured human cells, CPEB1 was shown to be assembled in stress granules 371 (Wilczynska et al., 2005). In contrast to Pum1 and Tdrd3, HuR, HuB and CPEB1 would 372 be constitutional components of RNA granules in mouse oocytes regardless of the 373 timing of translational activation of assembled mRNAs. 374 The tudor gene was identified in Drosophila as one of the maternal factors that 375 regulate embryonic development and fertility (Boswell and Mahowald, 1985). The tudor
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376 domain forms a b-barrel-like core structure that contains three short b-strands followed 377 by an a-helical region, and over 200 tudor domain-containing proteins have been 378 identified from many eukaryotes including fungi, plants and animals (Lasko, 2010; 379 Ponting, 1997). Many tudor domain-containing proteins harbor RNA-binding motifs, 380 chromatin-binding domains, or DNA-binding domains and have been shown to function 381 in diverse cellular processes such as histone modification, DNA damage response, RNA 382 splicing, small RNA pathway, and pi-RNA-mediated transposon silencing (Maurer- 383 Stroh et al., 2003; Siomi et al., 2010). The full length of Tdrd3 was identified on the 384 basis of its tudor domain and has been shown to possess several domains, which may be 385 involved in interaction with nucleic acid and/or proteins (Goulet et al., 2008; Linder et 386 al., 2008). Using cultured human cells, Tdrd3 was shown to assemble into stress 387 granules in response to several types of stress. In breast cancer cells, Tdrd3 was shown 388 to selectively regulate the translation of certain mRNAs, which may promote growth 389 and invasion of breast cancer tumors (Morettin et al., 2017). However, whether Tdrd3 390 functions in physiological conditions has remained unknown. 391 In this study, we identified Tdrd3 as a protein binding specifically to Emi2 mRNA 392 by the RNA pull-down assay followed by mass spectrometry analysis (Fig. 3D and 393 Table S1). We showed that the N-terminal region of Tdrd3 (1-473 aa) bound to the 394 3’UTR of Emi2 mRNA in in vitro UV cross-linking analysis (Fig. 4D), indicating that 395 this region contains the amino acid sequences responsible for the direct binding to 396 mRNA. In addition, this region is able to select the target mRNA because Tdrd3-N did 397 not bind to the 3’UTR of cyclin B1 mRNA (Fig. 4D). Our results provide the first 398 evidence that Tdrd3 directly binds to mRNA and has target specificity. In GV-stage 399 mouse oocytes, Tdrd3 formed particles and colocalized with Emi2 RNA granules (Fig. 400 5). Interference in Tdrd3 function by the anti-Tdrd3 antibody delayed but did not 401 completely inhibit Emi2 synthesis in meiosis II (Fig. 6). Knockdown of Tdrd3 did not 402 induce or accelerate the derepression of Emi2 mRNA translation (Fig. 6), suggesting 403 that Tdrd3 is not a repressor of translation. Rather, it seems to promote the translation of 404 Emi2 mRNA in meiosis II, since knockdown attenuated the synthesis of Emi2 in 405 meiosis II. Intriguingly, both interference in Tdrd3 function by the anti-Tdrd3 antibody 406 and knockdown of Tdrd3 did not affect the translation of cyclin B1 and Mad2 mRNA in 407 meiosis I (Fig. 6). These results are consistent with the results of the IP/RT-PCR, RNA 408 pull-down assay, UV cross-linking, and FISH analyses (Figs. 3, 4 and 5) and support the
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409 notion that the accurate timing of translational activation of mRNA is determined by 410 components specific to distinct RNA granules. 411 Super-resolution microscopy of Tdrd3 and Pum1 in GV-stage mouse oocytes showed 412 differences in the sizes and shapes of Tdrd3 particles and Pum1 aggregates (Fig. 5). 413 Pum1 was shown to form solid-like aggregates in human cultured cells and GV-stage 414 mouse oocytes (Shiina, 2019; Takei et al., 2020). We previously showed that highly 415 structured aggregates of Pum1 in a solid-like state ensures translational repression of 416 cyclin B1 and Mad2 mRNAs and that dissolution of the aggregates allows the 417 disassembly of cyclin B1 and Mad2 RNA granules, resulting in the translational 418 activation of these mRNAs (Takei et al., 2020). The quantitative analyses showed that 419 the size of Tdrd3 particles was smaller than that of Pum1 aggregates (Fig. 5). Moreover, 420 Tdrd3 particles appeared in a round shape (Fig. 5), which might reflect the formation of 421 liquid droplets induced by liquid-liquid separation (Shin and Brangwynne, 2017). 422 Thereby, Tdrd3 might form soluble particles and act in a way different from that of 423 Pum1. We are currently addressing this possibility. Interestingly, Tdrd3 particles seemed 424 to form clusters in the oocyte cytoplasm (Figs. 5 and S3) and these distribution patterns 425 were maintained in meiosis I (T. Kotani, unpublished observations/data not shown). The 426 highly organized complexes including Tdrd3 might promote compartmentalization of 427 the cytoplasm and maintain silencing of translation in meiosis I. After completion of 428 meiosis I, Tdrd3 promotes the Emi2 mRNA translation by an unknown mechanism. One 429 possible explanation is that Tdrd3 recruits Dazl and/or poly(A) polymerases such as 430 mGLD-2 after completion of meiosis I, both of which have been shown to accumulate 431 in meiosis I and to promote translation of many dormant mRNAs in mouse oocytes 432 (Chen et al., 2011; Nakanishi et al., 2006; Sousa Martins et al., 2016). 433 In conclusion, we demonstrated the existence of Tdrd3-mediated translational 434 control of Emi2 mRNA, through which oocytes promote the progression of meiosis II, 435 generating fertilizable oocytes. The highly ordered temporal translation of mRNAs 436 would be achieved by the assembly of components common and specific to distinct 437 RNA granules. These granules were spatially and temporally organized in the oocyte 438 cytoplasm. Recent studies utilizing systematic approaches have demonstrated that more 439 than one hundred proteins are assembled in stress granules and that stress granules 440 compose diversities; specific proteins in addition to constitutive proteins are assembled 441 by different types of stress (Aulas et al., 2017; Jain et al., 2016; Markmiller et al., 2018).
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442 Compositional differences in stress granules were also observed in different types of 443 cells such as fibroblasts and neuronal cells. Our results will contribute to an 444 understanding of the differences in function and regulation of these granules in many 445 types of cells besides oocytes. 446 447 MATERIALS AND METHODS 448 Preparation of ovaries 449 All animal experiments in this study were approved by the Committee on Animal 450 Experimentation, Hokkaido University. Mouse ovaries were dissected from 8- to 12- 451 week-old adult females and placed in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
452 Na2HPO4, and 2 mM KH2PO4, pH 7.2). For in situ hybridization, mouse ovaries were 453 fixed with 4% paraformaldehyde in PBS (4% PFA/PBS) overnight at 4˚C. For 454 immunoprecipitation analysis, mouse ovaries were homogenized with an equal volume 455 of ice-cold extraction buffer (EB: 100 mM β-glycerophosphate, 20 mM Hepes, 15 mM
456 MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 100 µM (p-amidinophenyl) methanesulfonyl 457 fluoride, and 3 µg/ml leupeptin, pH 7.5) containing 1% Tween 20 and 100 U/ml RNase 458 inhibitor (Invitrogen). After centrifugation at 5,000 rpm for 15 min at 4˚C, the 459 supernatant was collected and used for immunoprecipitation. For the RNA pull-down 460 assay, cytoplasmic extracts of Xenopus oocytes were prepared according to the 461 procedure of the RiboTrap kit (MBL International). Ovaries were dissected from adult 462 females and cut into small pieces in nuclease-free PBS. Xenopus ovaries were 463 homogenized with an equal volume of ice-cold CE Buffer (+) (MBL International) and 464 incubated for 10 min on ice. Then the extracts were diluted with Detergent Solution 465 (MBL International) and centrifuged at 3,000 g for 3 min at 4˚C. The supernatant was 466 transferred to new tubes and diluted with High-Salt Solution (MBL International). After 467 centrifugation at 12,000 g for 3 min at 4˚C, the supernatant was collected and used for 468 the RNA pull-down assay. 469 470 Production of antibodies 471 DNA sequences encoding a part of mouse Emi2 (residues 1-366) was amplified by PCR 472 and ligated into pET21 vector to produce a histidine (His)-tagged protein. The 473 recombinant protein was expressed in E. coli and purified by SDS-PAGE, followed by 474 electroelution in Tris-glycine buffer without SDS. The purified protein was dialyzed
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475 against 1 mM HEPES (pH 7.5), lyophilized, and used for injection into two mice. The 476 obtained antisera were affinity-purified with recombinant Emi2-His protein 477 electroblotted onto a membrane (Immobilon; EMD Millipore). 478 479 Collection of oocytes 480 For immunoblotting, PAT assay and injection experiments, GV-stage oocytes were 481 collected by puncturing ovaries with a needle in M2 medium (94.7 mM NaCl, 4.8 mM
482 KCl, 1.2 mM KH2PO4, 1.3 mM MgCl2, 5.6 mM glucose, 23.3 mM sodium lactate, 4
483 mM NaHCO3, 0.3 mM sodium pyruvate, 1.7 mM CaCl2, 21 mM HEPES, 0.1 mM 484 gentamicin, 4 mg/ml BSA, pH 7.5) supplemented with 10 µM milrinone (M2+). 485 Resumption of meiosis was induced by washing oocytes with M2 medium lacking 486 milrinone (M2-) four times and culturing at 37˚C in drops of M2- covered with paraffin 487 liquid. At the appropriate time points after resumption of meiosis, oocytes were 488 collected for immunoblotting and the poly(A) test (PAT) assay. For immunoblot 489 analysis, the collected oocytes were washed with PBS and extracted in 10 µl lithium 490 dodecyl sulfate (LDS) sample buffer (NOVEX) or SDS sample buffer (50 mM Tris- 491 HCl; pH 6.8, 2% sodium dodecyl sulfate, 6% β-mercaptoethanol, 10% glycerol, 0.1% 492 phenol Red). For the PAT assay, oocytes were extracted with Trizol regent (Life 493 Technologies) according to the manufacturer’s instructions. 494 495 PAT assay 496 Two µg of total RNA extracted from pools of 200 mouse oocytes was ligated to 0.4 µg
497 of P1 anchor primer (5’-P-GGT CAC CTT GAT CTG AAG C-NH2-3’) in a 10-µl 498 reaction using T4 RNA ligase (New England Biolab) for 30 min at 37℃. The ligase was 499 inactivated for 5 min at 92˚C. Eight µl of the reaction was used in a 20 µl reverse 500 transcription reaction using the SuperScript III First Strand Synthesis System with P1’ 501 primer (5’-GCT TCA GAT CAA GGT GAC CTT TTT-3’). Aliquots of the cDNA were 502 used as templates of the 1st RCR with primer sets of P1’ primer and mEmi2-forward 503 primer-1 (5’-ATG TCT GTG CGC TTA TCA CG-3’) or P1’ primer and mcyclin B1- 504 forward primer-1 (5’-CCA CTC CTG TCT TGT AAT GC-3’). Aliquots of the 1st PCR 505 reactions were used for the 2nd PCR with primer sets of P1’ primer and mEmi2-forward 506 primer-2 (5’-TCC CAT TGA TGA CAG ACG TTG ATT TAC TTC CCA CTT-3’) or P1’ 507 primer and mcyclin B1-forward primer-2 (5’-CCT GGA AAA GAA TCC TGT CTC-
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508 3’). The PCR products were resolved on 3% TAE gel. It was confirmed by cloning the 509 2nd PCR products and sequencing them that the increase in PCR product length was 510 due to elongation of the poly(A) tails. 511 512 Polysomal fractionation 513 Polysomal fractionation was performed according to the procedure described previously 514 (Masek et al., 2020; Takada et al., 2020). Briefly, 200 oocytes were treated with 100 515 µg/ml of cycloheximide (CHX) for 10 min and collected in 350 µl of lysis buffer (10 516 mM Hepes, pH 7.5; 62.5 mM KCl, 5 mM MgCl2, 2 mM DTT, 1% TritonX-100) 517 containing 100 µg/ml of CHX and 20 U/ml of Ribolock (Thermo Fisher Scientific). 518 After disruption of the zona pellucida with 250 µl of zirconia-silica beads (BioSpec), 519 lysates were centrifuged at 8,000 g for 5 min at 4˚C. Supernatants were loaded onto 10- 520 50% linear sucrose gradients containing 10 mM Hepes, pH 7.5; 100 mM KCl, 5 mM 521 MgCl2, 2 mM DTT, 100 µg/ml of CHX, Complete-EDTA-free Protease Inhibitor (1 522 tablet/100 ml: Roche) and 5 U/ml of Ribolock. Centrifugation was performed using an 523 Optima L-90 ultracentrifuge (Beckman) at 35,000 g for 65 min at 4˚C. Polysome 524 profiles were recorded using an ISCO UA-5 UV absorbance reader. Ten equal fractions 525 were collected from each sample and subjected to RNA isolation by Trizol reagent 526 (Sigma). Fractions 6 to 10 were taken for polysome-bound RNA. Furthermore, a library 527 was prepared using the SMART-seq v4 ultra low input RNA kit (Takara Bio). 528 Sequencing was performed by HiSeq 2500 (Illumina) as 150-bp paired-end. Reads were 529 trimmed using Trim Galore v0.4.1 and mapped to the mouse GRCm38 genome 530 assembly using Hisat2 v2.0.5. Gene expression was quantified as fragments per 531 kilobase per million (FPKM) values in Seqmonk v1.40.0. 532 533 Injection of MOs, mRNAs and antibodies 534 The sequences of antisense morpholino oligonucleotides (MOs) (Gene Tools, LLC) are 535 as follows: Emi2-3’UTR-MO, 5’-AT T TAT TT CT TAAAAGT CCCAA CAA-3’, that 536 specifically targets the terminal sequence of the Emi2 mRNA 3’UTR and Emi2-3’UTR 537 5mm-MO, 5’-AT T CAT T ACTTAATAGTCACATCAA-3’, that contains 5-nts 538 mismatches (underlines) and was used as a control. GV-stage oocytes were injected with 539 10 pl of a solution containing 0.1 mM Emi2-3’UTR-MO or Emi2-3’UTR 5mm-MO 540 using an IM-9B microinjector (Narishige) under a Dmi8 microscope (Leica) in M2
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541 medium. After being injected, the oocytes were cultured in M16 medium in an
542 atmosphere of 5% CO2 in air at 37˚C and used for immunoblotting or morphological 543 observation after fixation with 4% PFA for 1 h. 544 mRNAs encoding GFP and GFP-Emi2 were synthesized using the mMESSAGE 545 mMACHINE SP6 kit (Ambion) and dissolved in nuclease-free distilled water. Eight pl 546 of 1 µg/µl mRNAs was injected into GV-stage oocytes in M2+ using the IM-9B 547 microinjector under the Dmi8 microscope and washed four times with M2- to resume 548 meiosis. Similarly, 8 pl of 0.1 mg/ml anti-Tdrd3 antibody (Cell Signaling Technology; 549 D302G) or control IgG was injected into GV-stage oocytes and washed four times with 550 M2-. To knock down Tdrd3 by the Trim-Away system, 8 pl of a mixture containing 500 551 ng/µl RFP-Piranha mRNA (System Biosciences) and 0.1 mg/ml anti-Tdrd3 antibody 552 (Cell Signaling Technology; D302G) was injected into GV-stage oocytes and incubated 553 at 37˚C in M2+ for 2-3 h to allow Piranha protein expression. The oocytes were then 554 washed four times with M2- to resume meiosis. 555 556 Whole mount immunofluorescence 557 Oocytes were collected at the appropriate time points after incubating with M2- at 37˚C. 558 After fixation with 4% PFA in PBS for 1 h, the oocytes were permeabilized in
559 permeabilization buffer (0.3% BSA, 0.1% Triton-X, 0.02% NaN3 in PBS) for 20 min, 560 washed three times with blocking solution (0.3% BSA, 0.01% Tween 20 in PBS), and 561 incubated in blocking solution for 20 min at room temperature. The oocytes were then 562 incubated with anti-b-tubulin-Cy3 antibody (Sigma) for 1 h at room temperature. After 563 washing three times with blocking solution, the oocytes were mounted on slides with 564 VECTASHIELD Mounting Medium containing DAPI (Vector Laboratories, lnc) and 565 observed under an LSM 5 LIVE confocal microscope (Carl Zeiss). 566 567 Section in situ hybridization 568 Section in situ hybridization with the TSA Plus system (PerkinElmer) was performed 569 according to the procedure reported previously (Takei et al., 2018). In brief, the fixed 570 ovaries were dehydrated, embedded in paraffin, and cut into 7-µm-thick sections. The 571 digoxigenin (DIG)-labeled sense or antisense RNA probe for full-length Emi2 was 572 synthesized with a DIG-RNA-labeling kit (Roche Molecular Biochemicals). No signal 573 was detected with sense probes. The sections of mouse ovaries were hybridized with a
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574 hybridization mix containing 1 ng/µl of DIG-labeled RNA probe. After hybridization 575 and washing, the sections were incubated with anti-DIG-horseradish peroxidase (HRP) 576 antibody (1:500 dilution; Roche Molecular Biochemicals) for 30 min. The reaction with 577 tyramide-DNP, followed by reaction with the anti-DNP-alkaline phosphatase (AP) 578 antibody, and the detection of signals were performed according to the manufacturer’s 579 instructions. For FISH analysis, the samples were incubated overnight with anti-DNP– 580 Alexa Fluor 488 antibody (1:500 dilution; Molecular Probes) after reaction with 581 tyramide DNP. To detect nuclei, the samples were incubated with 10 µg/ml Hoechst 582 33258 for 10 min. 583 Double fluorescence in situ hybridization of cyclin B1 and Emi2 mRNAs was 584 performed as follows. Sections of mouse ovaries were hybridized with a fluorescein- 585 labeled cyclin B1 RNA probe and DIG-labeled Emi2 RNA probe. After hybridization 586 and washing, samples were incubated with anti-DIG-HRP antibody (1:500 dilution) for 587 30 min. After reaction with tyramide-DNP, the samples were incubated with anti-DNP- 588 Alexa Fluor 488 antibody (1:500 dilution) overnight. Before detection of the
589 fluorescein-labeled cyclin B1 RNA probe, the samples were incubated with 1% H2O2 in 590 PBS for 15 min for inactivating HRP. The reaction with tyramide-Cy3 (1:50 dilution in 591 1×Plus Amplification Diluent (PerkinElmer), followed by 1:100 dilution with DW) was 592 performed for 20 min. To detect nuclei, the sections were incubated with 10 µg/ml 593 Hoechst 33258 for 10 min and observed under the LSM 5 LIVE confocal microscope. 594 595 Section immunofluorescence 596 Section immunofluorescence was performed according to the procedure reported 597 previously (Takei et al., 2020). Briefly, ovary sections were microwaved for 10 min 598 (500 W) with 10 mM citric acid (pH 6.0). After incubation with a TNB blocking 599 solution (PerkinElmer) at room temperature for 1 h, the samples were incubated with 600 anti-TDRD3 antibody (Cell Signaling Technology; D302G) and/or anti-Pum1 (Bethyl; 601 A300-201A) antibody overnight at room temperature. The samples were then incubated 602 with anti-goat IgG-Alexa Flour Plus 555 antibody (Invitrogen) and/or anti-goat IgG- 603 Alexa Flour Plus 647 antibody (Invitrogen) at room temperature for 1 h. After staining 604 with Hoechst 33258, the samples were mounted and observed under an N-SIM super- 605 resolution microscope (Nikon). No signal was detected in the reaction without primary 606 antibodies. To simultaneously detect the protein and mRNA, the samples were
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607 immunostained as described above after detection of the Emi2 or cyclin B1 RNA probe 608 with tyramide-FITC (PerkinElmer, Inc.) in in situ hybridization analysis. The size and 609 roundness of signals were measured by ImageJ software. The creation of random dots 610 and measurements of the distances of Tdrd3 particles and randomly distributed dots 611 were performed by using ImageJ. 612 613 Immunoprecipitation followed by RT-PCR (IP/RT-PCR) 614 Eighty µl of mouse ovary extracts was incubated with 2 µg of anti-Pum1 (Bethyl; A300- 615 201A), anti-HuR (SANTA CRUZ; sc-5261) or anti-ELAVL2 (HuB) (Proteintech; 616 14008-1-AP) antibodies and protein G Mag sepharose (GE Healthcare) overnight at 617 4˚C. the same volume of IgG was used as a control. The samples were washed five 618 times with EB containing 1% Tween 20. An aliquot of the samples was used for RT- 619 PCR with primer sets specific to cyclin B1, mcyclin B1- forward primer-2 (5’-CCT 620 GGA AAA GAA TCC TGT CTC-3’) and mcyclin B1- reverse primer-1, specific to 621 Emi2, mEmi2-forward primer-2 (5’-TCC CAT TGA TGA CAG ACG TTG ATT TAC 622 TTC CCA CTT-3’) and Emi2-reverse primer (5’-GGG GAT ATG ACA TAG TAA AA- 623 3’), and specific to α-tubulin, mα-tubulin-forward primer (5’-CTT TGT GCA CTG GTA 624 TGT GGG T-3’) and mα-tubulin-reverse primer (5’-ATA AGT GAA AT G GGC AGC 625 TTG GGT-3’). 626 627 RNA pull-down assay and mass spectrometry 628 The RNA-binding assay was performed using the RiboTrap Kit (MBL International) 629 according to the manufacturer’s instructions. In brief, bromouridine (BrU)-labeled 630 RNAs of the 3’ UTR of mouse Emi2, Xenopus Emi2, and mouse cyclin B1 were 631 generated using the Riboprobe in vitro Transcription Systems kit according to the 632 manufacturer’s protocol (Promega). Anti-BrdU antibodies were conjugated with protein 633 A-sepharose beads overnight at 4℃ (GE Healthcare). Then the RNAs were bound to the 634 beads. Cytoplasmic extracts of Xenopus oocytes were transferred to tubes containing the 635 BrU-labeled RNAs conjugated with the beads for 2 h at 4℃. The samples were washed 636 with Wash BufferII, eluted with elution buffer (4% BrdU/DMSO solution in nuclease- 637 free PBS; MBL International), and subjected to SDS-PAGE. The SDS-PAGE gels were 638 stained by silver staining with the Silver Stain MS kit (Wako Pure Chemical Industries, 639 Ltd) to visualize the proteins associated with BrU-labeled RNAs. The bands were
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640 excised from the gel and subjected to a mass spectrometric analysis with Orbitrap Velos 641 Pro (Thermo Fisher Scientific). For protein identification, MASCOT 2.5.1 was used for 642 database searching against NCBInr_Xenopus laevis (updated on 05/09/2015, 17,456 643 sequences). The results from each run were filtered with the peptide confidence value, 644 in which peptides showing a false discovery rate (FDR) of less than 1% were selected. 645 In addition, proteins identical to the sequenced peptides with the rank1 values were 646 selected. The number of peptides identical to the proteins was more than one. We 647 counted the number of proteins that were pulled down with the mouse and Xenopus 648 Emi2 RNA probes as candidate proteins specifically binding to Emi2 mRNA, with all of 649 the RNA probes as candidates binding to both Emi2 and cyclin B1 mRNAs, and with 650 the mouse cyclin B1 RNA probe as candidates specifically binding to cyclin B1 mRNA. 651 652 UV-cross linking assay 653 The full length of Tdrd3 was amplified by PCR using Tdrd3-f1 (5’-TGG ATC CTA 654 TGG CCG AGG TGT CCG-3’) and Tdrd3-r1(5’-AT C GAT GTT CCG AGC T CG 655 AGG-3’). The N-terminal region of Tdrd3 (1-473 aa) was amplified by PCR using 656 Tdrd3-f1 and Tdrd3-r2 (5’-AT C GAT T GG CCT GT C ACA CT T-3’). The C-terminal 657 region of Tdrd3 (246-744 aa) was amplified by PCR using Tdrd3-f2 (5’-GGA TCC TAT 658 GGG TGG TGC CAG AAG TAA-3’) and Tdrd3-r1. The PCR products were inserted 659 into pGEM-T Easy vector (Promega). Sequences encoding the full length and parts of 660 Tdrd3 were cloned into pCS2-Flag-N or pCS2-Flag-C to produce Tdrd3 fused with Flag 661 at the N- or C-terminus of Tdrd3. mRNAs encoding Flag-Tdrd3, Flag-Tdrd3-N and 662 Flag-Tdrd3-C were synthesized with an mMESSAGE mMACHINE SP6 kit (Ambion), 663 and the resulting mRNAs (2 µg) were translated in 50 µl of rabbit reticulocyte lysate 664 (Promega). Flag-tagged proteins were purified by immunoprecipitation with anti-Flag 665 M2 antibody (Sigma) and Protein G Mag Sepharose (GE healthcare). The DIG-labeled 666 3’UTR of Emi2 and cyclin B1 RNA probes were synthesized with a DIG-RNA-labeling 667 kit (Roche Molecular Biochemicals). Two µg of probes was incubated with purified 668 Flag-tagged proteins for 15 min, and the reaction mixtures were irradiated twice in a 669 UV cross-linker (XL-1000, Funakoshi) at an energy setting of 86 mJ/cm2. 670 671 Immunoblotting 672 Mouse oocyte extracts were separated by SDS-PAGE with Bolt Bis-Tris Plus Gels
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673 (Novex), blotted onto an immobilon membrane using a Bolt Mini Blot Module (Novex), 674 and probed with anti-Emi2 (this study), anti-cyclin B1 (Abcam; V152), anti-γ-tubulin 675 (Sigma; T6557), anti-Tdrd3 (Cell Signaling Technology; D302G) and anti-Mad2 676 (Bethyl; A300-301A) antibodies. The crude extracts from mouse ovaries and rabbit 677 reticulocyte lysates and the immunoprecipitates were separated by SDS-PAGE, blotted 678 onto an immobilon membrane, and probed with anti-Pum1 (Bethyl; A300-201A), anti- 679 HuR (SANTA CRUZ; sc-5261), anti-ELAVL2 (HuB) (Proteintech; 14008-1-AP), anti- 680 FLAG (Sigma; F1804-1MG), anti-TDRD3 (Cell Signaling Technology; D302G) and 681 anti-DIG-AP (Roche) antibodies. The intensity of signals was quantified using ImageJ 682 software. 683 684 Acknowledgements 685 We thank Dr. K. Kobayashi for technical advice on super-resolution microscopy and S. 686 Kawamura for an initial experiment of the PAT assay. 687 688 Competing interests 689 The authors declare no competing or financial interests. 690 691 Author contributions 692 Conceptualization: N. Takei and T. Kotani. Investigation: N. Takei, K. Sato, Y, Takada 693 and R. Iyyappan. Resources: A. Susor and T. Yamamoto. Project administration: T. 694 Kotani. Writing - original draft: N. Takei and T. Kotani. Writing - review and editing: A. 695 Susor, T. Yamamoto and T. Kotani. 696 697 Funding 698 This work was supported by Grant-in-Aid for Scientific Research (16K07242 to T.K.) 699 from the Ministry of Education, Culture, Sports, Science and Technology, Japan and 700 was in part supported by a grant from Japan Society for the Promotion of Science 701 (JSPS) KAKENHI grant number JP16H06280. This work was partly performed in the 702 Cooperative Research Project Program of the Medical Institute of Bioregulation, 703 Kyushu University. 704 705
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706 References 707 Abrieu, A., Doree, M., and Fisher, D. (2001). The interplay between cyclin-B-Cdc2 708 kinase (MPF) and MAP kinase during maturation of oocytes. J. Cell Sci. 114, 709 257-267. 710 Anderson, P., and Kedersha, N. (2009). RNA granules: post-transcriptional and 711 epigenetic modulators of gene expression. Nat. Rev. Mol. Cell Biol. 10, 430-436. 712 Aulas, A., Fay, M.M., Lyons, S.M., Achorn, C.A., Kedersha, N., Anderson, P., and 713 Ivanov, P. (2017). Stress-specific differences in assembly and composition of 714 stress granules and related foci. J. Cell Sci. 130, 927-937. 715 Boswell, R.E., and Mahowald, A.P. (1985). tudor, a gene required for assembly of the 716 germ plasm in Drosophila melanogaster. Cell 43, 97-104. 717 Buchan, J.R., and Parker, R. (2009). Eukaryotic stress granules: the ins and outs of 718 translation. Mol. Cell 36, 932-941. 719 Chalupnikova, K., Solc, P., Sulimenko, V., Sedlacek, R., and Svoboda, P. (2014). An 720 oocyte-specific ELAVL2 isoform is a translational repressor ablated from 721 meiotically competent antral oocytes. Cell Cycle 13, 1187-1200. 722 Chen, J., Melton, C., Suh, N., Oh, J.S., Horner, K., Xie, F., Sette, C., Blelloch, R., 723 and Conti, M. (2011). Genome-wide analysis of translation reveals a critical 724 role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition. 725 Genes Dev. 25, 755-766. 726 Clift, D., McEwan, W.A., Labzin, L.I., Konieczny, V. , Mogessie, B., James, L.C., 727 and Schuh, M. (2017). A Method for the Acute and Rapid Degradation of 728 Endogenous Proteins. Cell 171, 1692-1706 e1618. 729 Colombrita, C., Silani, V. , and Ratti, A. (2013). ELAV proteins along evolution: back 730 to the nucleus? Mol Cell Neurosci. 56, 447-455. 731 Conti, M., and Franciosi, F. (2018). Acquisition of oocyte competence to develop as 732 an embryo: integrated nuclear and cytoplasmic events. Hum. Reprod. Update. 733 24:245-266. 734 Fujimura, K., Katahira, J., Kano, F., Yoneda, Y., and Murata, M. (2009). 735 Microscopic dissection of the process of stress granule assembly. Biochim. 736 Biophys. Acta. 1793, 1728-1737. 737 Gallouzi, I.E., Brennan, C.M., Stenberg, M.G., Swanson, M.S., Eversole, A., 738 Maizels, N., and Steitz, J.A. (2000). HuR binding to cytoplasmic mRNA is
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838 HuR-hnRNP interactions and the effect of cellular stress. Mol. Cell Biochem. 839 372, 137-147. 840 Polanski, Z., Ledan, E., Brunet, S., Louvet, S., Verlhac, M.H., Kubiak, J.Z., and 841 Maro, B. (1998). Cyclin synthesis controls the progression of meiotic 842 maturation in mouse oocytes. Development 125, 4989-4997. 843 Ponting, C.P. (1997). Tudor domains in proteins that interact with RNA. Trends 844 Biochem. Sci. 22, 51-52. 845 Richter, J.D. (2007). CPEB: a life in translation. Trends Biochem. Sci. 32, 279-285. 846 Sagata, N. (1996). Meiotic metaphase arrest in animal oocytes: its mechanisms and 847 biological significance. Trends Cell Biol. 6, 22-28. 848 Schmidt, A., Duncan, P.I., Rauh, N.R., Sauer, G., Fry, A.M., Nigg, E.A., and Mayer, 849 T.U. (2005). Xenopus polo-like kinase Plx1 regulates XErp1, a novel inhibitor of 850 APC/C activity. Genes Dev. 19, 502-513. 851 Setoyama, D., M. Yamashita, and N. Sagata. (2007). Mechanism of degradation of 852 CPEB during Xenopus oocyte maturation. Proc. Natl. Acad. Sci. USA. 104, 853 18001-18006. 854 Shiina, N. (2019). Liquid- and solid-like RNA granules form through specific scaffold 855 proteins and combine into biphasic granules. J. Biol. Chemi. 294, 3532-3548. 856 Shin, Y., and Brangwynne, C.P. (2017). Liquid phase condensation in cell physiology 857 and disease. Science. 357, eaaf4382. 858 Shoji, S., Yoshida, N., Amanai, M., Ohgishi, M., Fukui, T., Fujimoto, S., Nakano, 859 Y., Kajikawa, E., and Perry, A.C. (2006). Mammalian Emi2 mediates 860 cytostatic arrest and transduces the signal for meiotic exit via Cdc20. EMBO J. 861 25, 834-845. 862 Siomi, M.C., Mannen, T., and Siomi, H. (2010). How does the royal family of Tudor 863 rule the PIWI-interacting RNA pathway? Genes Dev. 24, 636-646. 864 Sousa Martins, J.P., Liu, X., Oke, A., Arora, R., Franciosi, F., Viville, S., Laird, 865 D.J., Fung, J.C., and Conti, M. (2016). DAZL and CPEB1 regulate mRNA 866 translation synergistically during oocyte maturation. J. Cell Sci. 129, 1271-1282. 867 Stebbins-Boaz, B., Hake, L.E., and Richter, J.D. (1996). CPEB controls the 868 cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is 869 necessary for oocyte maturation in Xenopus. EMBO J. 15, 2582-2592. 870 Suzuki, T., Suzuki, E., Yoshida, N., Kubo, A., Li, H., Okuda, E., Amanai, M., and
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871 Perry, A.C. (2010). Mouse Emi2 as a distinctive regulatory hub in second 872 meiotic metaphase. Development 137, 3281-3291. 873 Takada, Y., Iyyappan, R., Susor, A., and Kotani, T. (2020). Posttranscriptional 874 regulation of maternal Pou5f1/Oct4 during mouse oogenesis and early 875 embryogenesis. Histochem. Cell Biol. 154, 609-620. 876 Takei, N., Nakamura, T., Kawamura, S., Takada, Y., Satoh, Y., Kimura, A.P., and 877 Kotani, T. (2018). High-sensitivity and high-resolution in situ hybridization of 878 coding and long non-coding RNAs in vertebrate ovaries and testes. Biol. Proced. 879 Online 20, 6. 880 Takei, N., Takada, Y., Kawamura, S., Sato, K., Saitoh, A., Bormann, J., Yuen, W.S., 881 Carroll, J., and Kotani, T. (2020). Changes in subcellular structures and states 882 of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs. J. 883 Cell Sci. 133, jcs249128. 884 Tay, J., R. Hodgman, and Richter, J.D. (2000). The control of cyclin B1 mRNA 885 translation during mouse oocyte maturation. Dev. Biol. 221, 1-9. 886 Tung, J.J., D.V. Hansen, K.H. Ban, A.V. Loktev, M.K. Summers, J.R. Adler, 3rd, 887 and P.K. Jackson. (2005). A role for the anaphase-promoting complex inhibitor 888 Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of 889 Xenopus eggs. Proc. Natl. Acad. Sci. USA. 102, 4318-4323. 890 Tung, J.J., Padmanabhan, K., Hansen, D.V., Richter, J.D., and Jackson, P.K. 891 (2007). Translational unmasking of Emi2 directs cytostatic factor arrest in 892 meiosis II. Cell Cycle 6, 725-731. 893 Wada, T., Hara, M., Taneda, T., Qingfu, C., Takata, R., Moro, K., Takeda, K., 894 Kishimoto, T., and Handa, H. (2012). Antisense morpholino targeting just 895 upstream from a poly(A) tail junction of maternal mRNA removes the tail and 896 inhibits translation. Nucleic Acids Res. 40, e173. 897 Wilczynska, A., Aigueperse, C., Kress, M., Dautry, F., and Weil, D. (2005). The 898 translational regulator CPEB1 provides a link between dcp1 bodies and stress 899 granules. J Cell Sci. 118, 981-992. 900 Winata, C.L., and Korzh, V. (2018). The translational regulation of maternal mRNAs 901 in time and space. FEBS lett. 592, 3007-3023. 902 Yang, Y., Yang, C.R., Han, S.J., Daldello, E.M., Cho, A., Martins, J.P.S., Xia, G., 903 and Conti, M. (2017). Maternal mRNAs with distinct 3' UTRs define the
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904 temporal pattern of Ccnb1 synthesis during mouse oocyte meiotic maturation. 905 Genes Dev. 31, 1302-1307. 906 907
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908 Figure legends 909 Fig. 1. Polyadenylation-dependent translational control of Emi2 mRNA defines the 910 timing of Emi2 accumulation in mouse oocytes. (A, left) Immunoblotting of Emi2, 911 cyclin B1 and γ-tubulin in oocytes at 0, 8 and 18 h after resumption of meiosis. (right) 912 Quantitative analysis (means ± SD; n = 3). The statistical significance was analyzed by 913 the Tukey-Kramer test. * P < 0.05, **P < 0.01. (B) Time course of the PAT assay of 914 Emi2 (upper) and cyclin B1 (lower) mRNAs after resumption of meiosis. Similar results 915 were obtained from three independent experiments. Note that poly(A) tails of a fraction 916 of cyclin B1 mRNA were shortened in MII oocytes, possibly due to the induction of 917 meiosis in vitro. (C) RNA-seq-based gene expression values (FPKM) for Emi2 in GV- 918 and MII-stage oocytes (means ± SD; n = 3). t-test: * P < 0.05. (D, upper) Sequences of 919 the 3’UTR of Emi2 mRNA that are targeted by Emi2-3’UTR-MO. Emi2-5mm-MO 920 contains 5 mismatches (red). (D, lower) Immunoblotting of Emi2 and γ-tubulin in 921 oocytes not injected (-) and injected with Emi2-5mm-MO and Emi2-3’UTR-MO at 28 h 922 after resumption of meiosis. (E) Immunofluorescence of β-tubulin (red) in oocytes 923 injected with Emi2-5mm-MO and Emi2-3’UTR-MO at 28 h and 48 after resumption of 924 meiosis. DNA is shown in blue. (F) Immunofluorescence of β-tubulin (red) in oocytes 925 injected with GFP or GFP-Emi2 mRNA at 18 h after resumption of meiosis. Right 926 figure shows a bright field. DNA is shown in blue. (G) Percentage of metaphase I- or 927 metaphase II-stage oocytes at 18 h after resumption of meiosis. The numbers in 928 parentheses indicate the total numbers of oocytes analyzed. PB, polar body. Bars: 20 929 µm. 930 931 Fig. 2. Emi2 mRNA forms granules that are different from cyclin B1 RNA 932 granules. (A) Expression of Emi2 mRNA in the mouse ovary. A mouse ovary section 933 hybridized with the Emi2 antisense probe (left) and sense probe (right). (B) FISH 934 analysis of Emi2 mRNA in GV-stage oocytes. (C) FISH analysis of Emi2 (green) and 935 cyclin B1 (red) mRNAs in GV-stage oocytes. (insets) Enlarged views of the boxed 936 region. (D) FISH analysis of oocytes at 0, 4, 18 h after resumption of meiosis. (insets) 937 Enlarged views of the boxed region. (E) The numbers of RNA granules per 100 µm2 in 938 individual oocytes at 0, 4, and 18 h were counted (mean ± SD). The numbers in 939 parentheses indicate the total numbers of oocytes analyzed. The statistical significance 940 was analyzed by the Tukey-Kramer test. **P < 0.01. fc, follicle cells; GV, germinal
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941 vesicle; PB, polar body. Bars, 10 µm in A, B and D, 20 µm in C. 942 943 Fig. 3. Specific and common interactions of RNA-binding proteins with Emi2 944 mRNA. (A-C) Immunoblotting of mouse ovary extracts before IP (Initial) and IP with 945 control IgG (IgG) or anti-Pum1 (α-Pum1) (A), anti-HuR (α-HuR) (B) and anti-HuB (α- 946 HuB) (C) antibodies and semi-quantitative RT-PCR amplification for Emi2, cyclin B1 947 and α-tubulin transcripts. Graphs show the results of quantitative analyses of RT-PCR 948 (means ± SD; n = 3). t-test: *P < 0.05, **P < 0.01. (D) Venn diagram depicts the 949 number of proteins isolated as proteins interacting with 3’UTR sequences of Emi2 (red) 950 and cyclin B1 (blue) with the number of overlapping proteins. Keratin and ribosomal 951 proteins were excluded. (E) Gene ontology analysis of genes enriched in proteins 952 isolated as proteins interacting with 3’UTR sequences of Emi2 (upper), Emi2 and cyclin 953 B1 (middle) and cyclin B1 (lower). 954 955 Fig. 4. Tdrd3 is expressed in mouse oocytes and interacts with the 3’UTR of Emi2 956 mRNA. (A) Schematic diagrams of Tdrd3, Tdrd3-N and Tdrd3-C. (B) RT-PCR 957 amplification for Tdrd3 mRNA in the ovary and oocytes and immunoblotting of Tdrd3 958 in extracts of the mouse ovary and oocytes at GV and MII stages. (C) UV-cross linking 959 assay of the 3’UTR of Emi2 and cyclin B1 mRNA with Flag-CPEB1. The upper panel 960 shows immunoprecipitated Flag-CPEB1. The lower panel shows DIG-labeled probes 961 that interact with Flag-CPEB1. An asterisk shows IgG of anti-Flag antibody. (D, left) 962 UV-cross linking assay of the 3’UTR of Emi2 and cyclin B1 mRNA with Tdrd3-N-Flag. 963 The upper panel shows immunoprecipitated Tdrd3-N-Flag. The lower panel shows 964 DIG-labeled probes that interact with Tdrd3-N-Flag. An asterisk shows IgG of anti-Flag 965 antibody. (D, right) Quantitative analysis for the intensities of RNA probes (means ± 966 SD; n =3). 967 968 Fig. 5. Emi2 RNA granules contain Tdrd3. (A, left) Immunofluorescence of Tdrd3 in 969 GV-stage oocytes. DNA is shown in blue. (right) An enlarged view of the boxed region. 970 Similar results were obtained from three independent experiments. (B) Double 971 immunofluorescence of Tdrd3 (red) and Pum1 (green) in GV-stage oocytes. (insets) 972 Enlarged views of the boxed region. DNA is shown in blue. Similar results were 973 obtained from three independent experiments. (C) Average distances (µm) between one
31 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
974 Tdrd3 particle and 5 neighboring particles and average distances of random dots (n = 50 975 from three independent experiments). t-test: * P < 0.05. (D) Average sizes (µm2) of 976 Tdrd3 and Pum1 (n = 90 from three independent experiments). t-test: * P < 0.05. (E) 977 Roundness of Tdrd3 and Pum1 (n = 90 from three independent experiments). t-test: * P 978 < 0.05. (F) Immunofluorescence of Tdrd3 (red) and FISH analysis of Emi2 (upper, 979 green) or cyclin B1 (lower, green) mRNAs. Arrows indicate Tdrd3 overlapped with 980 Emi2 RNA granules. (G) Immunofluorescence of Pum1 (red) and FISH analysis of 981 Emi2 (upper, green) or cyclin B1 (lower, green) mRNAs. Arrows indicate Pum1 982 overlapped with cyclin B1 RNA granules. GV, germinal vesicle. Bars: 10 µm in A and 983 B, 2 µm in C and D. 984 985 Fig. 6. Knockdown of Tdrd3 attenuates Emi2 synthesis in meiosis II but not cyclin 986 B1 and Mad2 synthesis in meiosis I. (A) Immunoblotting of Emi2, cyclin B1 and 987 Mad2 in oocytes injected with IgG or anti-Tdrd3 antibody at 0, 8, 12, and 18 h after 988 resumption of meiosis. (B) Quantitative analysis for the amount of Emi2 in (A) (means 989 ± SD; n =3). t-test: * P < 0.05. (C) Immunoblotting of Tdrd3 and γ-tubulin in oocytes 990 not injected (-) and injected (+) with Piranha mRNA and anti-Tdrd3 antibody at 18 after 991 resumption of meiosis. (D) Immunofluorescence of β-tubulin in oocytes injected with 992 Piranha mRNA and IgG (left), Piranha mRNA and anti-Tdrd3 antibody (middle), or 993 Piranha mRNA, anti-Tdrd3 antibody and GFP-Emi2 mRNA at 14 h after resumption of 994 meiosis. Lower panels show enlarged views of the boxed region. DNA is shown in blue. 995 PB, polar body. Bars: 20 µm. (E) Percentage of oocytes with a bipolar or monopolar 996 spindle or those that entered interphase in oocytes injected with Piranha mRNA and IgG 997 (P + IgG), Piranha mRNA and anti-Tdrd antibody (P + α-Tdrd3), or Piranha mRNA, 998 anti-Tdrd3 antibody and GFP-Emi2 mRNA (P + α-Tdrd3 + Emi2) at 14 h after 999 resumption of meiosis. (F) Immunoblotting of Emi2, cyclin B1 and Mad2 in oocytes 1000 injected with Piranha mRNA and IgG (P + IgG) or Piranha mRNA and anti-Tdrd3 1001 antibody (P + α-Tdrd3) at 0, 8, 12, and 18 h after resumption of meiosis. (G) 1002 Quantitative analyses for the amounts of Emi2, cyclin B1 and Mad2 in (F) (means ± 1003 SD; n =3). t-test: * P < 0.05, ** P < 0.01.
32 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
cyclin B1 A (GV) (MI) (MII) Emi2 (kDa) ** ** 0 h 8 h 18 h ** * * -- Emi2 1 1
0.5 0.5 49 -- -- cyclin B1 Relative amount
49 -- Relative amount -- γ-tubulin 0 0 0 h 8 h 18 h 0 h 8 h 18 h (GV) (MI) (MII) (GV) (MI) (MII)
(GV) (PMI) (MII) B (bp) C 0 h 4 h 18 h 8 400 -- *
300 -- Emi2 6
200 -- 4 300 -- mRNA (FPKM) mRNA
cyclin B1 2 200 -- Emi2 f f Polysomal occupation o 100 -- 0 GV MII
D E 28 h 48 h Emi2 3’UTR Poly(A) signal No injection Emi2-5mm-MO Emi2-3’UTR-MO Emi2-3’UTR-MO 5’-ATATTTTGATGTTTTAAATAAAGAATTTTCAGGGTTGTT poly(A)-3’ PB PB ｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜｜ PB Emi2-3’UTR-MO 3’-ATTTATTTCTTAAAAGTCCCAACAA-5’ Emi2-5mm-MO 3’-ATTCATTACTTAATAGTCACATCAA-5’
28 h
(kDa) (-)
-- Emi2
49 -- -- γ-tubulin
F PB G Metaphase II Metaphase I (39) (53) (97) GFP 100%100
80%80
60%60
40%40
20%20 Percentage of oocytes
0 GFP-Emi2 0% (-) GFP GFP-Emi2
Figure. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A C GV GV fc fc GV
Antisense Sense Emi2
B GV
cyclin B1
fc fc Merge
D E 0 h (GV) 4 h (PMI) 18 h (MII) Emi2 Emi2 GV PB cyclin B1 (10) (8) (6) (3) (3) (3) 14 30 ** ** 12 ** ** 25 10 20 8 15 6 PB cyclin B1 10 4 Number Number of granules Number Number of granules
2 5
0 0 0h 4h 18h 0h 4h 18h (GV) (PMI) (MII) (GV) (PMI) (MII)
Figure. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
IP 25 Initial IgG α-Pum1 IgG A (kDa) ** 20 α-Pum1 98 -- Pum1 (bp) 15 Emi2 300 -- 10 300 -- cyclin B1 5 Relative amount -tubulin 300 -- α 0 Emi2 cyclin B1 α-tubulin IP 40 Initial IgG α-HuR IgG B (kDa) ** 34 -- HuR 30 α-HuR (bp) ** 300 -- Emi2 20 cyclin B1 300 -- 10 Relative amount 300 -- α-tubulin 0 Emi2 cyclin B1 α-tubulin IP (kDa) C Initial IgG α-HuB 43 -- 200 * IgG HuB α-HuB (bp) 150 Emi2 300 -- 100
300 -- cyclin B1 50 ** Relative amount 300 -- α-tubulin 0 Emi2 cyclin B1 α-tubulin
D E Emi2 DNA replication Proteins interacting with: translation RNA processing protein complex assembly cellular amino acid metabolic process regulation of actin filament cell adhesion microtubule-based process 0 1 2 3 Enrichment score 71162 65 Emi2 + cyclin B1 RNA stabilization translation protein transport RNA processing nucleoside metabolic process ncRNA processing RNA localization 3’ UTR of 3’ UTR of protein complex assembly Emi2 mRNA cyclin B1 mRNA glycogen metabolic process gene silencing by miRNA nucleotide biosynthetic process meiotic cell cycle response to cytokine 0 1 2 3 4 Enrichment score cyclin B1 eye development neurogenesis negative regulation of cell communication regulation of actin filament regulation of Rho protein signal transduction regulation of neurogenesis protein complex assembly positive regulation of developmental process 0 1 2 3 Enrichment score
Figure. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B Tudor Ovary Oocyte OB- (bp) 1 743 DUF fold UBA FMRP EBM 300 -- Tdrd3 Tdrd3 1 473 Oocyte Tdrd3-N (kDa) 246 743 Ovary GV MII Tdrd3-C 95 -- Tdrd3 72 --
C D
** IP: α-Flag IP: α-Flag 1
α -Flag -- Flag-CPEB1 α -Flag -- Tdrd3-N-Flag * * IB IB 0.5 α-DIG -- RNA probe α-DIG -- RNA probe
Emi2-3’UTR: + + -- Emi2-3’UTR: + + -- Relative intensity probe of intensity RNA Relative cyclin B1-3’UTR: -- + + cyclin B1-3’UTR: -- + + 0 Emi2-3’UTR: + + -- UV: - + - + UV: - + - + cyclin B1-3’UTR: -- + + UV: - + - +
Figure. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
Tdrd3 Tdrd3 GV GV
Pum1
Merge
C DE
1 3 0.3 ＊ ＊ ＊
) 0.8 2 2 0.2 0.6
0.4 Roundness 1 Area ( μ m 0.1
proteins proteins ( μ m) 0.2
0 0 0 Distance Distance to neighboring Tdrd3 Random Tdrd3 Pum1 Tdrd3 Pum1
F G Tdrd3 Emi2 RNA Merge Pum1 Emi2 RNA Merge
Tdrd3 cyclin B1 RNA Merge Pum1 cyclin B1 RNA Merge
Figure. 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431574; this version posted February 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C IgG Injected: IgG α-Tdrd3 Emi2 α-Tdrd3 -- + + : Piranha mRNA (GV) (MI) (PMII) (MII) (GV) (MI) (PMII) (MII) 1.2 0 h8 h12 h 18 h 0 h 8 h 12 h 18 h - + - + : α-Tdrd3 (kDa) 1 * Emi2 (kDa) 0.8 Tdrd3 cyclin B1 0.6 49 -- 49 -- 0.4 γ-tubulin 28 -- Mad2 Relative amount 0.2
0 0 h 8 h 12 h 18 h (GV) (MI) (PMII)(MII) D E Piranha mRNA + α-Tdrd3 Piranha mRNA + IgG Piranha mRNA + α-Tdrd3 + GFP-Emi2 Bipolar spindle 14 h 14 h PB PB 14 h PB 14 h PB Monopolar spindle Interphase
(19) (31) (18) 100
80
60
40
20
Percentage of oocytes 0
DAPI DAPI DAPI DAPI β-tubulin β-tubulin β-tubulin β-tubulin
F G P + IgG P + α-Tdrd3
Injected: P + IgG P + α-Tdrd3 Emi2 cyclin B1 Mad2 1.2 1.2 1.2 (GV) (MI) (PMII) (MII) (GV) (MI) (PMII) (MII) ** * 0 h 8 h 12 h 18 h 0 h 8 h 12 h 18 h 1 1 1 * Emi2 0.8 0.8 0.8 (kDa) 0.6 0.6 0.6 49 -- cyclin B1 0.4 0.4 0.4 28 --
Mad2 Relative amount 0.2 Relative amount 0.2 Relative amount 0.2
0 0 0 0 h 8 h 12 h 18 h 0 h 8 h 12 h 18 h 0 h 8 h 12 h 18 h (GV) (MI) (PMII)(MII) (GV) (MI) (PMII)(MII) (GV) (MI) (PMII)(MII)
Figure. 6