Bridge Integrator 2, a New Specific Target for Improving Reproductive Life Span through RPS6 and NNT

Female ovary is the earliest degenerated organ and it faces distinct medical disadvantages that impair primordial follicle reserve and oocyte quality. Herein, we found that bridge integrator 2 (Bin2) was predominant within mouse ovaries and oocytes, and global-knockout of Bin2 improved both female fertility and oocyte quality with healthy physiology in mice. Ovarian quantitative proteomics and phosphomics showed that Bin2 knockout specifically decreased only p-RPS6 of mTOR pathway; meanwhile, it increased nicotinamide nucleotide transhydrogenase (NNT), the free-radical detoxifier, over 6-fold. Mechanically, phosphorylation at Thr423 & Ser424 translocated membrane Bin2 into cytoplasm to phosphorylate RPS6, while p-RPS6 bound 42-95 bp NNT UTR to inhibit NNT translation. We then synthesized a peptide (BPP) to mimic Bin2 inhibition, and found that 3-week BPP injection improved primordial follicle reserve and oocyte quality in aging or chemotherapeutics-treated mice. In all, Bin2 inhibition improve both primordial follicle reserve and oocyte quality without discernible side effects. oocyte mitochondrial defects are partially prevented showed that was much higher family unknown function, 241-505 AA); SDS-PAGE and commassie staining showed that both purified proteins had good purity and expected size. H. In-vitro phosphorylation and western blot showed that Bin2-DUF can phosphorylate RPS6 more than an equal amount of Bin2-BAR. I. In-vitro dose-dependent phosphorylation assay and western blot showed that Bin2-DUF could phosphorylate RPS6 much more strongly than Bin2-BAR. blot translation. 5’ UTR seven shorter or four longer regions in-vitro transcribed single-strand RNA of these regions. N and O. SDS-PAGE, commassie staining, and western blot showed that RPS6 (N) and Bin2 (O) proteins had phosphorylated portions. P and Q. Protein-RNA UV crosslinking and gel shift assay that p-RPS6 prefer to bind short region NNT instead of other UTR regions or Quantification crosslinking that p-Bin2 did of I. Continuous follicle counting showed that both total follicles and PMFs significantly increased in the BPP-treated group. J and K. Western blot and quantification showed that p-RPS6 significantly decreased in the BPP-treated group. L and M. Immunostaining and quantification showed that the NNT level within the oocytes significantly increased in the BPP-treated group. N and O. We examined how well BPP could recover ovarian function in the aging mice at the transcriptional level. RNA sequencing showed that at the fold threshold of |log2(2-month/9-month)| ≥ 2, there were 1858 DEGs (differentially-expressed genes) between 2-month ovaries and 9-month ovaries; at the fold threshold of |log2(9-month-BPP/9-month)| ≥ 2, there were 1138 DEGs (differentially-expressed genes) between 9-month-BPP ovaries and 9-month ovaries; and between these two clusters of DEGs, 622 DEGs overlapped. P. KEGG analysis of functionally-annotated DEGs showed that multiple follicle survival, oocyte quality, or oocyte steriodgenesis-related pathways are among the top 10 enriched signaling pathways. Scale bar for B, 200 µm; scale bar for H and L, 100 µm. * indicates p ˂ 0.05. substances. Upon transition from normal (young) ovaries to Bin2 -KO or -inhibited normal (young) ovaries, p-Bin2 is downregulated and conduced to p-RPS6 downregulation, NNT upregulation, and ROS downregulation, which eventually improve PFR and oocyte quality. Upon transition from normal (young) ovaries to aging & POF ovaries, p-Bin2 is upregulated and conduced to p-RPS6 upregulation, NNT downregulation, and ROS upregulation, which eventually decrease PFR and oocyte quality. Upon transition from aging & POF ovaries to Bin2 -KO or -inhibited aging & POF ovaries, higher p-Bin2 is downregulated close to normal level and conduced to higher p-RPS6 downregulated close to normal level, lower NNT upregulated close to normal level, and higher ROS downregulated close to normal level, which eventually revive PFR and oocyte quality.


INTRODUCTION
Reproductive health is important for the overall health not only of the individual self, but also of the next generation. Due to distinct physiology, women's ovaries, the core reproductive organ containing oocyte-producing primordial follicles (PMFs) and sex hormone-producing somatic cells, start to degenerate at least 20 years earlier than men's testes. Moreover, the ovary is more vulnerable than the testis to a variety of intrinsic and extrinsic adverse factors (1, 2). Additionally, chemotherapy against gynecological tumors, such as ovarian cancers (OC), always severely reduce ovarian primordial follicle reserve (PFR) and lower oocyte quality and thus cause premature ovarian failure (POF) (3,4). An ideal medication would be capable of improving ovarian PFR as well as oocyte quality, and meanwhile, not interfering with normal physiology; however, such a medication has yet to be realized.
For maintaining ovarian PFR, researchers have found that the complicated "dialogue" between oocytes, granulosa cells, theca cells, and other cells is crucial for ovarian function. Many factors (ligands) and receptors play important roles in the "dialogue" process (5-8); and one of the critical downstream signal mediators of these upstream receptors is MTOR, especially mTORC1. mTORC1 targets plenty of downstream kinases to promote a variety of important processes such as protein synthesis, metabolism, ribosomal synthesis, and transcription, thus promoting follicle survival and activation (9-12). Based on these, for translational medicine, inhibition of the activating factors of the MTOR pathway can theoretically inhibit the activation of PMFs, thereby prolonging follicle reserve during the natural aging process. For example, inhibition of cyclooxygenase-1 (cox-1) can prolong the life of postnatal follicles (13). Cox-1 up-regulation is IL4-dependent and regulated by Fes-Akt-mTOR (13). Sirt1 activator SRT1720 could also improve follicle reserve and prolong ovarian life by inhibiting the MTOR pathway (14). As a direct inhibitor of MTOR, rapamycin has been shown to preserve the primordial follicle pool and thereby increases ovarian lifespan in young and aging female mice (15). Caloric restriction can also protect follicle reserve by inhibiting the MTOR pathway (16). However, despite the positive roles of selective inhibition of mTOR pathway activators, several concerns still remain.
Firstly, kinases within mTOR pathway are mostly universally active and important in all tissues, inhibition of these kinases could disrupt normal biological processes and substantially hurt overall body health. For example, rapamycin is also an immunosuppressant and might lead to adverse metabolic reactions (17). Secondly, patients usually have medium to high levels of drug-related adverse reactions (such as diarrhea and nausea) to these chemicals (18). Thirdly, inhibition of these mTOR activators are "double-edged swords," in that an excessive inhibition could reduce follicle survival and lower oocyte quality since they are critical for these aspects (5-12).
Besides, other PFR-improving strategies beyond inhibition of mTOR activators, such as activating the SIRT1/FOXO1 signaling through hADSC-secreted HGF and bFGF (19) or up-regulating erk phosphorylation through Ptk2b knockout (20), could not specifically target ovaries as well and carry the risk of tumorigenesis (21,22).
For improving oocyte quality, although the physiological level of ROS (reactive oxygen species) is essential for follicular rupture and oocyte maturation, excessive ROS generation under disadvantageous circumstance such as psychological stress, aging, or chemotherapy is detrimental (23). Elevated ROS could conduce to enzyme inactivation, lipid peroxidation, ATP depletion, mitochondrial disruption, and meiotic spindle defects, finally result in decreased oocyte quality, apoptosis and reduced fertility (24). Therefore, many translational researches supported that oocyte quality could be improved by reducing ROS. For example, Nicotinamide Mononucleotide (NMN) supplementation eliminated accumulated ROS to suppress apoptosis in aged oocytes (25,26). CoQ10 supplementation decreased ROS levels and prevented abnormal mitochondrial distribution and in oocytes of obese mice (27). BAT transplantation could also improve oocyte quality of aging mice (28). However, the upper and most of other anti-oxidants, and BAT transplantation could not increase PFR, and non-enzymatic anti-oxidants are less efficient than enzymatic ROS detoxifiers such as SOD (superoxide dismutase) and Gpx (glutathione peroxidase) (23,25,26). It has been reported that supplement of melatonin (29), quercetin (30), or resveratrol (31) could improve both PFR and oocyte quality by reducing ROS and apoptosis. However, melatonin can inhibit the hypothalamic-pituitary-gonadal axis or directly act on the ovary to reduce the content of androgens, estrogens and progesterone (32). Quercetin is reported to be mutagenic in some studies (33).
Resveratrol has to be daily-taken for as long as 12 months in mice (about 40 years in human) to show significant increase in PFR (31). In addition, the clinical efficacy of all these treatments remains controversial.
In the current study, we found that a novel target, Bin2, might be a critical regulator of both PFR and oocyte quality. Since Bin2 is fairly dominant in ovaries and oocytes, target inhibition of Bin2 to prolong female fertility and improve oocyte quality did not affect normal physiology.

Bin2 is rich in ovaries & oocytes and essential for primordial follicle activation
Many important cancer-related genes are essential for ovarian function and female fertility. We herein have called these cancer/ovary genes, in concert with reported cancer/testis genes. Bin2 (Bridge integrator 2, gene ID 668218) was also named BRAP1 (breast cancer-associated protein 1). It has been reported that in diverse human cancers, including breast cancer and ovarian cancer, Bin2 has over 40 enhanced post-translational modification sites, among which most are phosphorylation sites (https://www.phosphosite.org/proteinAction?id=9451&showAllSites=true), suggesting that Bin2 might be over-activated through hyper-phosphorylation to promote tumorigenesis. We primarily showed that Bin2 overexpression in human epithelial ovarian cancer (EOC) cells significantly up-regulated p-RPS6 of the mTOR pathway while not altering p-Akt (Fig. 1A), while Bin2 knockdown in human epithelial ovarian cancer (EOC) cells significantly down-regulated p-RPS6 while also not altering p-Akt ( Fig. 1B). p-RPS6 over-activation is a major cause of premature activation of the primordial follicle pool (34). Bin2's function in female fertility is as yet unknown.
We first found that Bin2 protein was the richest in ovaries, as compared with other major tissues (Fig. 1C), and Bin2 protein level sharply increased as initial follicle recruitment occurred (Fig. 1D). Bin2 mRNA level in oocytes was much higher than the other two Bin family members, Bin1 and Bin3 (Fig. 1E) and remained constant during meiosis (Fig. 1F). Moreover, Bin2 protein was more dominant in oocytes than in granulosa cells ( Fig. 1G and 1H). These indicated that Bin2 might have important functions within ovaries, and primarily within oocytes.
To uncover its function, we used the cas9 technique to delete the whole exon 9 (83 bases) of Bin2 genome to obtain Bin2 global knockout mice ( Fig 1I). Western blot showed that Bin2 protein completely disappeared in the knockout ovaries (Fig. 1J).
Surprisingly, Bin2 knockout did not affect ovary weight, body weight, and ovary/body weight ratio; moreover, Bin2 knockout did not affect oocyte maturation or zygote development (Suppl. Fig. 1A-E). While the 60-week-long fertility assay showed that both the cumulative litter number per mouse and the average litter size significantly increased (Fig 1K-N). Further continuous follicle count showed that from 6 months old, both the total follicle number and the primordial follicle number significantly were significantly higher in Bin2-knockout ovaries ( Fig. 1O-R). Additionally, Bin2 knockout did not affect major immunity or inflammation indexes including C-reactive protein (CRP), CD4, and IL6 within plasma (Suppl Fig. 2A and 2B) and ovaries (Suppl. Fig.   2C and 2D) when compared with the control. These results indicated that Bin2 depletion could significantly improve primordial follicle survival and increase fertility without affecting the overall health of the female mice.

Phosphorylation at Thr423 & Ser424 translocated Bin2 into cytoplasm to activate RPS6
From the above results, Bin2 might be a novel target for the safe augmentation of primordial follicle reserve and ovarian lifespan. Since Bin2 is highly conserved between diverse mammalian species, including mouse and human, investigating the mechanism by which Bin2 functions in female mice might ultimately contribute to the improvement of ovarian performance in human females. We used TMT-labeled quantitative phosphomics to systematically identify differentially phosphorylated 8 proteins (≥ 1.2 -fold or ≤ 0.833 -fold) between control and Bin2-knockout 3-week-old ovaries and carefully observed pathways known to be involved in primordial follicle survival and activation and oocyte quality maintenance-for example, mTOR pathway ( Fig. 2A). We found that only RPS6 of the mTOR pathway had significantly decreased phosphorylation ( Fig. 2B and 2C), which was verified by Western blot and immunofluorescence ( Fig. 2D-F in the c-terminal domain of unknown function (DUF domain). We also found that these two residues and the adjacent residues are highly conserved between humans, rats, mice, rhesus monkeys, and Cynomolgus macaques ( Fig. 3A and 3B), so we hypothesized that these two residues might be the key residues for the activation and translocation of Bin2. We generated a phosphorylation antibody using a core phospho peptide "QSKRAASIQR(pThr) (pSer)A" and verified its specificity through Bin2 siRNA and corresponding non-phospho peptide "QSKRAASIQRTSA" (Fig 3C   and 3D). Immunostaining showed that Bin2 was enriched on the oocyte membrane, while p-Bin2 was enriched within the nucleus at the GV stage and within spindles during meiosis, which was quite similar to p-RPS6 (Fig 3E and 3F, Suppl. Fig. 3).
Next, to further verify the relationship between Bin2 and RPS6, we injected the non-phospho peptide "QSKRAASIQRTSA" into the GV oocytes and speculated that this peptide could compete with the endogenous Bin2, thereby decreasing the phosphorylation of endogenous Bin2. We found that p-Bin2 level significantly decreased, while p-RPS6 level also significantly decreased ( Fig 3G). In all, these results supported the conclusion that p-Bin2 could specifically activate (phosphorylate) RPS6 without affecting other components of the mTOR pathway.

In vitro characterization of Bin2 phosphorylation activity on rps6
We attempted to directly verify the relationship between Bin2 and RPS6 and the key roles of Thr423 and Ser424 on Bin2 activation. We used the Bac-to-bac system to express full-length wild-type Bin2 (Bin2-WT) and a Thr423 and Ser424 to Ala423 and Ala 424 mutant Bin2 (Bin2-AA) (Fig. 4A). We also expressed and purified RPS6 from E. coli. We found that the primary advantage of bacteria-derived RPS6 was that it was fully inactive (non-phosphorylated). Firstly, Bin2-WT can phosphorylate RPS6 more effectively than an equal amount of Bin2-AA (Fig. 4B). Next, in vitro dose-dependent phosphorylation assay showed that Bin2-WT could phosphorylate RPS6 much more strongly than Bin2-AA ( Fig. 4C and 4D). Immunostaining on bacularvirus-infected sf9 cells showed that Bin2-WT localized both on the membrane and within the cytoplasm, while Bin2-AA almost exclusively localized on the membrane; accordingly, p-RPS6 intensity in Bin2-WT bacularvirus-infected sf9 cells was significantly higher than in Bin2-AA bacularvirus-infected sf9 cells ( Fig. 4E and   4F).
Next, we used the Bac-to-bac system to express and purify two different motifs of Bin2, BAR (1-240 AA) and DUF (domain of unknown function, 241-505 AA), and examined the difference between them (Fig. 4G). Firstly, we found that Bin2-DUF could phosphorylate RPS6 more effectively than an equal amount of Bin2-BAR ( Fig.   4H). Secondly, in vitro dose-dependent phosphorylation assay showed that Bin2-DUF could phosphorylate RPS6 much more strongly than Bin2-BAR ( Fig. 4I and 4J).
These results further supported the conclusion that Bin2 phosphorylation at Thr423 and Ser424 is the primary mechanism for activating RPS6.

Bin2 knockout promotes nicotinamide nucleotide transhydrogenase translation through down-regulating p-RPS6
TMT-labelled quantitative proteomics (proteomics were used to normalize the phosphomics) between control and Bin2-KO 3-week-old ovaries showed that, among the differentially expressed gross proteins ( ≥ 1.2 -fold or ≤ 0.833 -fold), nicotinamide nucleotide transhydrogenase (NNT) was most up-regulated (> 6-fold, Fig. 5A and 5B, arrow pointed). We have reliably verified the NNT increment through blot ( Fig. 5C and   5D). In the meantime, NNT mRNA remained unchanged ( Fig. 5E and 5F). Moreover, RPS6 knockdown also up-regulated NNT protein level while keeping NNT mRNA level ( Fig. 5G and 5H). NNT regenerated NADP into NADPH and could act as free-radical detoxifier (35), and it was shown that elevated NNT improved oocyte quality and developmental competence (36). Thus, we hypothesized that Bin2 knockout could improve oocyte quality through NNT. Live dye staining showed that the ROS level significantly decreased ( Fig. 5I and 5J), while the JC-1 level significantly increased ( Fig. 5K and 5L) within Bin2-knockout oocytes. These indicated that Bin2 knockout could even improve oocyte quality.
Next, we tried to address whether p-Bin2 or p-RPS6 binds the translation-regulating regions of NNT to inhibit its translation. We found that NNT 5' UTR (untranslated region) are very conserved between multiple species including humans, rats, mice, rhesus monkeys, and Cynomolgus macaques (Supple. Fig. 4).
We subdivided NNT 5' UTR into seven shorter or four longer regions (Fig. 5M). We used Bin2 and RPS6 purified from sf9 cells, since these proteins were verified to have phosphorylated portions ( Figure 5N and 5O). Protein-RNA UV crosslinking and gel shift assay showed that p-RPS6 preferred to bind short regions 3 and 4, from 42 to 95 bp of NNT 5' UTR, instead of other UTR or CDS regions ( Fig. 5P and 5Q, arrow pointed), whereas p-Bin2 did not bind any shorter or longer regions ( Fig. 5R and 5S).
These results suggested that p-Bin2 negatively regulates NNT translation through p-RPS6, while p-RPS6 negatively regulates NNT translation by directly binding NNT 5' UTR region.

Inhibition of Bin2 phosphorylation in vivo prolongs ovarian lifespan in aging mice
As stated above, female ovarian function and fertility generally begins to drop at 35 years of age. The existing strategies for prolonging ovarian lifespans in mice have demonstrated several side effects. Since Bin2 knockout increased fertility without discernible side effects, we proposed that Bin2 might be a novel target for safely prolonging ovarian function and female fertility. We planned to use 8-month-old aging mice as models to mimic 35-year-old women (Fig. 6A), and we tested whether inhibiting Bin2 phosphorylation through the non-phospho peptide "QSKRAASIQRTSA" could largely mimic the effects of Bin2 knockout in mice.
To facilitate the permeation of this peptide in vivo, we fused PEP1, the known cell-penetrating peptide (37), to this peptide and renamed it as BPP (Bin2-penetrating peptide, Fig. 6B). We firstly verified that FITC-labeled BPP can efficiently enter ovaries ( Fig. 6C) or in vitro-cultured oocytes and A2780 cells (Suppl. Fig. 5A and 5B), and that both p-Bin2 and p-RPS6 significantly decreased within ovaries (Fig. 6D). We also showed that BPP had typical dose-dependent and time-dependent dynamics within the plasma (Suppl. Fig. 6A and 6B). Next, after a 3-week-long BPP injection cycle (once a day) on 8-month-old aging mice, fertility assay from 36 weeks old to 63 weeks old showed that the number of cumulative pups significantly increased ( Fig.   6E-G). Continuous follicle counting showed that total follicles and PMFs, and primary follicles ( Fig 6H and 6I, Supple. Fig. 7A and 7B) all increased. Accordingly, p-RPS6 also significantly decreased ( Fig. 6J and 6K), while NNT level within the oocytes significantly increased ( Fig. 6L and 6M). In addition, major organ weight (Suppl. Fig.   8A and 8B) and major blood biochemical indexes (Suppl. Fig. 9) assays did not change after Bin2 inhibition. These results indicated that ovary function in aging mice have been improved by Bin2 inhibition without discernible side effects.
Finally, we examined how well BPP could revive ovarian function in the aging mice at the transcriptional level. RNA sequencing showed that at the fold threshold of |log2(2-month/9-month)| ≥ 2, there were 1858 DEGs (differentially-expressed genes) between 2-month ovaries and 9-month ovaries; at the fold threshold of |log2(9-month-BPP/9-month)| ≥ 2, there were 1138 DEGs (differentially -expressed genes) between 9-month-BPP ovaries and 9-month ovaries; and between these two clusters of DEGs, 622 DEGs overlapped, suggesting that BPP injection could significantly recover the 9-month ovarian profile, bringing it close to the 2-month ovarian profile. (Fig 6N and 6O). KEGG analysis showed that multiple follicle survival, oocyte quality, or oocyte steriodgenesis-related pathways were among the top 10 enriched signaling pathways ( Fig 6P).

Inhibiting Bin2 phosphorylation in vivo protects ovaries from chemotherapy-induced premature ovarian failure
It is known that chemotherapy can cause premature ovarian failure (POF); one of the major mechanisms of this is mTOR pathway over-activation. Therefore, mTOR inhibitors could rescue ovarian function and fertility (13,14,17). However, mTOR-inhibiting chemicals also have multiple side effects (17,18). We wanted to test whether BPP could also rescue chemotherapy-induced POF safely and effectively.
Western blot showed that a harsh, three-week-long cyclophosphamide (CTX) treatment cycle significantly elevated p-RPS6, while BPP treatment reduced p-RPS6 level close to the control level (Fig. 7A). Moreover, CTX treatment significantly decreased oocyte NNT level, while BPP injection recovered NNT level close to the control ( Fig. 7B and 7C). Consequently, fertility assay from 4 months old to 9 months old showed that CTX treatment almost induced complete infertility, while BPP significantly recovered fertility, although still less than that of the control (Fig. 7D-F).
Continuous follicle counting showed that CTX treatment sharply decreased numbers of PMFs and growing follicles, while BPP significantly up-regulated follicle numbers, although still less than those of the control ( Fig. 7G and 7H). These results indicated that BPP could effectively preserve ovarian primordial follicle pool and simultaneously maintain oocyte quality during chemotherapy.  Fig. 4). And notably, although these regions are highly variable among humans, cynomolgus monkeys, macaque monkeys, mice, and rats, the peptide sequences around Thr423 and Ser424 are highly conserved (Supple. Fig.   10, shadowed in the rectangle), suggesting that the conserved activation/inactivation mechanism might rely on these two residues. On the other side, human Bin2 is also called BRAP-1 (Breast cancer-associated protein 1) and has 40 reinforced post-translational modification sites in diverse human tumors, suggesting that its over-activation may promote cell proliferation. In the present study, we found that Bin2 knockout specifically down-regulated p-RPS6 of the mTOR pathway. RPS6

DISCUSSION
hyper-phosphorylation is closely related to follicular over-activation (34) [43][44][45]. In the present study, we showed that Bin2 might be a novel kinase that directly phosphorylates RPS6 (Fig. 4). Thus, Bin2 knockout or inhibition is predicted to moderately down-regulate p-RPS6. On the other side, RPS6 itself was also essential for primordial follicle survival; excessive RPS6 suppression or Rps6 knockout caused loss of PMFs (45), so the extent of RPS6 reduction must be moderate to ensure both the survival and increase of PMFs. In our study, Bin2 knockout or inhibition appeared to cause an optimum p-RPS6 reduction, which is the critical base of the duet function of Bin2.
The main function of NNT (nicotinamide nucleotide transhydrogenase) is to regenerate NADP into NADPH and maintain the mitochondrial redox balance (35); NNT deficiency causes vascular ROS production and exacerbates atherosclerotic plaque development (46). On the other side, NNT levels decrease as aging and mitochondrial NAD levels also decrease in somatic cells, while NNT overexpression could restore NAD levels and promote the reprogramming of aged somatic cells (47).
In our study, Bin2 knockout and inhibition both greatly increased NNT, which might be able to protect oocyte quality even in cases in which p-RPS6 is harshly reduced.
Moreover, we discovered that p-Bin2 regulates NNT translation through p-RPS6, and thus it is likely p-RPS6 that directly binds the 42-95 bp NNT 5' UTR region to inhibit NNT translation. Therefore, we provided a clear model for how Bin2 knockdown or inhibition can improve oocyte quality through p-RPS6 and NNT.
In all, we showed that Bin2 knockout or inhibition could have a duet effect (Fig. 8).
At the post-translational level, p-Bin2 specifically activated p-RPS6 of mTOR; thus, p-Bin2 inhibition improved the PFR by specifically and moderately down-regulating p-RPS6. At the translational level, p-RPS6 binds to 42-95 bp of NNT 5' UTR mRNA to inhibit NNT translation; thus, p-Bin2 inhibition up-regulated NNT protein levels to improve oocyte quality. Since Bin2 is fairly predominant in ovaries & oocytes, the effect of p-Bin2 inhibition might be limited primarily in ovaries & oocytes. Therefore, compared with the current strategies, Bin2 could be a novel, specific, and safe target for improving PFR and oocyte quality (Fig. 8, Supple. Fig. 11).

AVAILABILITY OF DATA AND MATERIAL
The data that support the findings of this study are available from the corresponding author upon reasonable request.  2014 20 (23)  Thr423 & Ser424 and the adjacent residues are highly conserved between humans, rats, mice, rhesus monkeys, and cynomolgus macaques. C and D. We hypothesized that Thr423 & Ser424 are the key residues for the activation and translocation of Bin2. We generated a phosphorylation antibody using a core phosph peptide "QSKRAASIQR(pThr)(pSer)A" and verified its specificity through Bin2 siRNA and corresponding non-phosph peptide "QSKRAASIQRTSA." E. Immunofluorescence showed that Bin2 was enriched on oocyte membrane, while p-Bin2 was enriched within spindles during meiosis. F. Immunofluorescence showed that p-Bin2 was enriched within the nucleus of GV oocytes, similar to p-RPS6. G. We injected the non-phosph peptide "QSKRAASIQRTSA" into GV oocytes and found that the p-Bin2 level significantly decreased, while the p-RPS6 level also significantly decreased. Scale bar, 20 µm. western blot showed that Bin2-DUF can phosphorylate RPS6 more than an equal amount of Bin2-BAR. I. In-vitro dose-dependent phosphorylation assay and western blot showed that Bin2-DUF could phosphorylate RPS6 much more strongly than Bin2-BAR. J.

Figure 5. Bin2 knockout promotes NNT translation through down-regulating p-RPS6.
A and B. We used TMT-labeled quantitative proteomics to systematically identify differentially expressed gross proteins between control and Bin2-knockout, 3-week-old ovaries (≥ 1.2 fold or ≤ 0.833 fold). Among the differentially expressed gross proteins, nicotinamide nucleotide transhydrogenase (NNT) was most up-regulated (> 6 fold, red-arrow pointed). C and D. Western blot and quantification verified that the NNT protein level in the Bin2-knockout group increased more than 6-fold compared to control. E and F.  RNA sequencing showed that at the fold threshold of |log2(2-month/9-month)| ≥ 2, there were 1858 DEGs (differentially-expressed genes) between 2-month ovaries and 9-month ovaries; at the fold threshold of |log2(9-month-BPP/9-month)| ≥ 2, there were 1138 DEGs

Knockout Mice
Animal experimental procedures in our study were all approved by the Animal

Ovarian Hematoxylin-Eosin Staining and Ovary Follicle Counting
Ovaries were obtained, washed and fixed in 10% buffered formalin or 4% PFA overnight, embedded in paraffin, continuously sectioned at 5 µm thickness, then stained with hematoxylin and eosin. The follicle stages were classified according to Pederson's standard. Only follicles with visible nuclei were counted. In brief, an oocyte surrounded by a single layer of flattened or cubical granulosa cell was defined as a primordial or primary follicle; an oocyte surrounded by more than one layer of cuboidal granulosa cells without visible antrum was defined as a secondary follicle; and a follicle possessing a clearly antral space containing follicular fluid was defined as antral follicle. The number of follicles counted per ovary was used for statistical analysis.

Identification of Bin2 Phosphorylation Site and Generation of p-Bin2-specific Antibody
Generally, the phosphorylated part of a protein is about 0.01-0.1%, making it almost impossible to use oocytes for identification of phosphorylation sites. Therefore, we used NIH3T3 cells instead, then generated a phospho-specific antibody and verify the antibody in oocytes. We used five IP reactions, each of which employed 2.5 x 10 6 , and the whole process of antibody production and purification was accomplished by Zoonbio Biotechnology (Nanjing, Jiangsu, China). A short phospho peptide QSKRAASIQR(pThr)(pSer)AC ("C" at the C-term is an extra residue for conjugation) was synthesized and injected into rabbits for the serum production. The phospho-specific antibody was purified from the serum through a column filled with phospho peptide-conjugated resin and then absorbed through a column filled with non-phospho peptide (QSKRAASIQRTSA)-conjugated resin to remove any residual non-phospho-specific antibody.

Proteins with Bac-to-bac System
EGFP-tagged Bin2-WT (wild type), Bin2-AA (T423A, S424A), Bin2-BAR (BAR domain of Bin2), Bin2-DUF (DUF domain of Bin2), and TagRFP-tagged rps6 proteins were cloned and expressed through a Bac-to-bac system (Thermo Fisher). In brief, rpm until OD600 reached 1.0. Next, 0.2 mM IPTG was added into the medium to induce the recombined RPS6 expression overnight at 16 °C, 220 rpm. The next morning, the E.coli was spun down, washed once with cold PBS, lysed, and purified as descried above.

In Vitro Phosphorylation Assays
BRB80 with 10% glycerol, 1 mM ATP and 5 mM DTT was used for the in vitro phosphorylation assays. Appropriate amounts of Bin2 proteins and RPS6 were incubated at RT for 20 minutes. Finally, the reaction was subjected to western blot.

TMT-Labeling Quantitative Proteomics and Phosphomics
About two hundred three-week-old ovaries per repeat (about 200 mg), two repeats for WT and Bin2-KO respectively, were sent to Hangzhou Jingjie Co.
(Hangzhou, China). In brief, ovaries were cracked and the supernatant was digested by trypsin into peptides. Then the peptides from individual samples were isobaric-mass tagged by TMT 6 -126, 127, 128, and 129, respectively, according to the manufacturer's protocol for TMT kit/iTRAQ kit (Thermo Fisher). Next, TMT-labeled tryptic peptides were fractionated by high pH reverse-phase HPLC using a Thermo Betasil C18 column, then peptide fractions were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC for identification of peptides (for proteomics) or phosphorylation sites (for phosphomics). The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8).

Assay of Mitochondrial Transmembrane Potential
Oocytes were incubated at 37 °C for 20 min with the fluorescent potentiometric indicator JC-1 (Cat#: 40706ES60, Yeasen, Shanghai, China) diluted 1:200, then washed twice with PBS and added to droplets (50 µl) of culture medium. Images of green fluorescence (JC-1 as monomers at low membrane potentials) and red fluorescence (JC-1 as aggregates at higher membrane potentials) were captured using confocal microscopy as above. Mitochondrial depolarization is indicated by a 38 decrease in the red/green fluorescence intensity ratio.

Detection of ROS Generation
The ROS Assay Kit (Cat#: S0033, Beyotime, Beijing, China) was used to detect ROS generation in oocytes. In brief, oocytes were incubated with a dichlorofluorescein diacetate (DCFH-DA) probe for 20 min at 37 °C in the dark, washed, and mounted on slides for confocal imaging.

RNA Sequencing and Analysis
RNA samples were collected from mouse ovaries. RNA isolation, library construction, and RNA sequencing (RNA-seq) were carried out by the Beijing Genomics Institute following standard protocols. The library products were sequenced using a BGISEQ-500. Standard bioinformatics analysis was performed by the Beijing Genomics Institute. For gene expression analysis, the significance of the differential expression genes was defined by the bioinformatics service of BGI according to the combination of the absolute value of |log2(treated/control)| ≥ 2 and q value < 0.001. All original sequence datasets have been submitted to the database of NCBI Sequence Read Archive (SRA) under accession number.

RNA-Protein Crosslinking and Gel Shift Assay
To determine whether p-Bin2 or p-RPS6 binds the 5'UTR region, and to meticulously analyze which sub-region of the 5'UTR is preferentially bound, we used two different scales of subdivision ( Figure 5M). For one scale, we sub-divided the whole 5' UTR of NNT into seven sub-regions (each are 20-30 bps long); for another scale, we sub-divided the whole 5' UTR of NNT into four sub-regions (each are 40-50 bps long).
Short single-strand RNAs were produced by using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA) according to the manufacturer's instructions.
In brief, for each single-strand RNA, a pair of synthesized complementary single-stranded DNA oligonucleotides were first annealed to form a double-stranded DNA template (Supple . Table 3). Subsequently, the single-stranded RNA was