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

<strong>Abstract</strong> 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 <em>Bin2</em> improved both female fertility and oocyte quality with healthy physiology in mice. Ovarian quantitative proteomics and phosphomics showed that <em>Bin2</em> 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 &amp; 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. <strong>Supplementary Dataset Legends</strong> <strong>Supplementary Dataset 1.</strong> Related to all fertility assays. WT mating male mice were monthly rotated between cages according to this random allocation table. <strong>Supplementary Dataset 2.</strong> Related to figure 2A–C. This excel file contains three sheets: The “All identified phospho sites” sheet includes all site-phosphorylation values and related identified information from two repeats of WT and Bin2-knockout PND-21 ovaries. The “Bin2-KO vs WT ≥ 1.2 up” sheet includes all differential site-phosphorylation values and related information with 1.2-fold up-regulation. The “Bin2-KO vs WT ≤ 0.83 down” sheet includes all differential site-phosphorylation values and related information with 0.83-fold down-regulation. <strong>Supplementary Dataset 3.</strong> Related to figures 2F, figure 5A and 5B. This excel file contains three sheets: The “All identified proteins” sheet includes all protein expression values and related identified information from two repeats of WT and Bin2-knockout PND-21 ovaries. The “Bin2-KO vs WT ≥ 1.2 up” sheet includes all differentially-expressed protein (DEP) values and related information with 1.2-fold up-regulation. The “Bin2-KO vs WT ≤ 0.83 down” sheet includes all differentially-expressed protein (DEP) values and related information with 0.83-fold down-regulation. <strong>Supplementary Dataset 4.</strong> Related to figure 2G. This excel file contains one sheet: “All gene expression-FPKM &amp; Log2” includes FPKM and Log2 values of all identified genes and related information from three repeats of PND-21 WT and Bin2-KO mouse ovaries. To avoid the illegal calculation of value “0,” we added a minimal value “0.001” to all original values (we have verified that this did not alter any differential trends). <strong>Supplementary Dataset 5.</strong> Related to figure 6N-P. This excel file contains four sheets: The “All gene expression FPKM &amp; Log2" sheet includes FPKM and Log2 values for all identified genes and related information from three repeats of the 2-month (2M), 9-month (9M), and 9-month + BPP (9M-BPP) groups. The “|Log2(Ave-2M)vs(Ave-9M)| ≥2” sheet includes all genes and related information with two-fold differential expression between the 2M and 9M groups. The “|Log2(Ave-9M-BPP)vs(Ave-9M)| ≥2” sheet includes all genes and related information with two-fold differential expression between the 9M-BPP and 9M groups. The “2Mvs9M-BPPvs9M overlap” includes all genes and related information that overlapped between “|Log2(Ave-2M)vs(Ave-9M)| ≥2” and “|Log2(Ave-9M-BPP)vs(Ave-9M)| ≥2”. To avoid the illegal calculation of value “0,” we added a minimal value “0.001” to all original values (we have verified that this did not alter any differential trends).