SUN-115 Elucidating the Molecular Mechanisms of Action of MKRN3 in the Regulation of Pubertal Timing

Abstract D. Franssen: None. A. Mancini: None. J. Haase: None. K. Corr: None. L. Naule: None. I. Landa: None. R.S. Carroll: None. A. Abreu: None. U.B. Kaiser: None. Background and Objectives: Loss-of-function mutations in MKRN3 are the most frequent genetic cause of central precocious puberty. MKRN3, or makorin ring finger protein 3, belongs to the makorin protein family, which is distinguished by zinc-finger motifs implicated in RNA binding and E3 ubiquitin ligase activity. Our laboratory has demonstrated, using both in vitro and in vivo models, that MKRN3 has an inhibitory action on the hypothalamic-pituitary-gonadal (HPG) axis that controls puberty and reproduction. In Mkrn3 KO mice, the levels of neurokinin B (NKB), a stimulator of the HPG axis, are increased in the hypothalamus. Recently, we identified an interaction between MKRN3 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a key factor in post-transcriptional regulation of gene expression and in neuronal plasticity. IGF2BP1 is also known to be regulated by LIN28B, which is associated with the timing of puberty in genome-wide association studies. Taken together, these data support the hypothesis that MKRN3, IGF2BP1 and LIN28B may work in conjunction to regulate NKB levels, and thereby pubertal development. Methods and Results: In HEK293 cells co-transfected with HA-MKRN3 and GFP-IGF2BP1, we found that the interaction of MKRN3 and IGF2BP1 is mediated by RNA, as their interaction was abrogated by RNase treatment. Using RNA co-immunoprecipitation, we found that MKRN3 and IGF2BP1 bind to the same RNA target, TAC3 mRNA (encoding NKB). The level of this protein, NKB, was significantly decreased when co-expressed in HEK293 cells together with wild-type MKRN3, compared to NKB alone (reduced to 27 ± 16 % of baseline, P < 0.05, N=3), while this decrease was abrogated if MKRN3 was mutated in its ubiquitin ligase motif (52 ± 23 % of baseline, NS, N=3). Moreover, we found that the decline in NKB in the presence of MKRN3 resulted from proteasome-mediated degradation. Proteasome inhibition led to a decrease of NKB, both in the presence and in the absence of MKRN3, likely through aggrephagy (i.e., degradation of aggregated proteins by the autophagosome) that occurs in response to proteotoxic conditions. However, if aggrephagy was inhibited by chloroquine, the reduction of NKB occurred only if MKRN3 was present. If both pathways - aggrephagy and proteasome - were inhibited, the decrease of NKB in the presence of MKRN3 was prevented. Lastly, we also found that co-expression of MKRN3 with LIN28B resulted in a decline in LIN28B protein levels in vitro, compared to expression of LIN28B alone. Conclusion: We have identified a novel molecular mechanism of action of MKRN3. MKRN3 interacts with IGF2BP1 via TAC3 mRNA and induces the degradation of the TAC3 encoded protein, NKB, via the ubiquitin-proteasome pathway. In addition, MKRN3 induces a decrease in LIN28B levels, which may modulate the MKRN3-IGF2BP1 pathway, as LIN28B is known to repress the production of let-7 microRNA, which targets IGF2BP1. Sunday, June 2, 2024