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Background and Objectives Human fertility is attained following puberty due to finely orchestrated events driven by hypothalamic release of the excitatory neuropeptide neurokinin B (NKB), which in turn stimulates the release of kisspeptin and then GnRH to activate the downstream pituitary-gonadal axis. Our group has identified that loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3) cause central precocious puberty (CPP). Mkrn3 is highly expressed in the hypothalamus of both male and female mice in early postnatal life, then declines prior to the onset of puberty. Mutations in MKRN3 E3 ubiquitin ligase domain, identified in patients with CPP, result in reduced auto-ubiquitination. However, the mechanisms of action of MKRN3 and its targets remain largely unknown. We hypothesized that MKRN3 inhibits the reproductive axis through interactions with hypothalamic substrates, targeting them to degradation pathways. Methods and Results Using interactome analysis and candidate approaches in vitro, we identified poly(A) binding protein cytoplasmic 1 and 4 (PABPC1 and 4) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as MKRN3 interactors, consistent with reports from other research groups. Additionally, our proteomic analysis revealed that Igf2bp1 levels are increased in the hypothalamus of Mkrn3 knockout (KO) mice versus wild type animals. Remarkably, we found that MKRN3-IGF2BP1 interaction is mediated by RNA, as their interaction was abrogated by RNase treatment. However, an MKRN3 missense mutation associated with CPP, p.C364F, within the E3 ubiquitin ligase domain, did not affect the RNA mediated interaction. Preliminary data from RNA co-IP, aiming to identify putative RNA targets, suggested that MKRN3 and IGF2BP1 bind to TAC3 mRNA. We further identified an increase in Nkb protein levels in the hypothalamus of Mkrn3 KO mice. We also showed that NKB is a substrate of MKRN3 E3 ubiquitin ligase activity in vitro by demonstrating increased ubiquitination of NKB in cells co-transfected with expression vectors encoding MKRN3 and NKB and treated with proteasome inhibitor. Additionally, MKRN3 overexpression resulted in degradation of NKB protein by western blot analysis, and alteration of NKB intracellular localization by immunofluorescence. These effects were impaired by MKRN3 missense mutations (p.C340G and p.C364F) in the E3 ubiquitin ligase motif, suggesting a pathophysiological mechanism of MKRN3 mutations in CPP. Conclusions We identified that MKRN3 interacts with PABPC1, PABPC4 and IGF2BP1 and targets NKB to degradation. We showed that NKB degradation was mediated by the MKRN3 E3 ubiquitin ligase domain, as mutations within this motif abrogated NKB degradation. However, this mutation did not affect MKRN3 RNA-mediated binding to IGF2BP1, highlighting that multiple domains of MKRN3 may contribute to its actions. Here, we propose a mechanism by which MKRN3, interacting with PABPCs and IGF2BP1 and targeting NKB for degradation, may act to inhibit the reproductive axis. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 a.m. |
