Fig 2: MKRN3 interacts with IGF2BP1.(A) Interactome map of key protein interactions with MKRN3. The network includes MKRN3 detected by different purification methods. Circle sizes indicate the CompPASS interaction score. Green circles indicate interaction identified in HEK293 cells, pink circles in SH-SY5Y cells, and orange circles in both cell lines. The oval nodes represent different clusters of prey proteins. (B) Co-IP analysis of MKRN3 and IGF2BP1 interaction. HEK293T cells were transiently transfected with HA-MKRN3, GFP-IGF2BP1, or both. Lysates were immunoprecipitated using anti-HA antibody. Both input and co-IP fractions were immunoblotted using anti-IGF2BP1 or anti-HA antibodies. The immunoblot demonstrates that IGF2BP1 is co-immunoprecipitated by anti-HA antibodies when coexpressed with HA-MKRN3.

Fig 3: Comparison of transcriptomes of MKRN3-KO and MKRN3-WT hiPSC-derived hypothalamic ARC neurons reveals differences in expression of genes that control hypothalamic neuronal development and plasticity.(A) Volcano plot comparing Benjamini-Hochberg–adjusted (BH-adjusted) P values against fold-change, showing transcripts that are differentially expressed between MKRN3-KO and MKRN3-WT hypothalamic ARC neurons (genes downregulated in MKRN3-KO neurons in blue, genes upregulated in MKRN3-KO neurons in orange, and genes not significantly different in gray). The analysis was performed using a BH adjusted P value cutoff of 0.05 and a log2 fold-change ratio cutoff of 1. Three representative genes more highly expressed in MKRN3-KO are shown. SLIT1, slit guidance ligand 1; SLIT2, slit guidance ligand 2; NRCAM, neuronal cell adhesion molecule. (B) Heatmap of the top 50 genes that are the most differentially expressed in MKRN3-KO 1 compared with MKRN3-WT 1 hypothalamic neurons. Each column represents 1 differentiation protocol. (C) Dot plot of Gene Ontology (GO) terms enriched between MKRN3-WT and MKRN3-KO hypothalamic neurons. The size of the dots represents the number of genes in the GO term, and the color gradient indicates the adjusted P value, using the BH method. (D–F) Heatmaps of differentially expressed genes (DEGs) in 3 significantly enriched pathways: (D) extracellular matrix (ECM) organization, (E) axon guidance, and (F) synapse organization, in MKRN3-KO compared with MKRN3-WT hypothalamic neurons. (G) Heatmap for a subset of DEGs between MKRN3-WT and MKRN3-KO hypothalamic neurons that have been associated with age at menarche in GWAS. (H) Relative SLIT1, SLIT2, and NRCAM mRNA levels in MKRN3-WT 1, MKRN3-WT 2, MKRN3-KO 1, and MKRN3-KO 2 hypothalamic neurons (n = 3 differentiation protocols per group). Data are presented as the mean ± SEM values. Statistics were performed using 2-way ANOVA, followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01.

Fig 4: Mkrn3 deletion in mice is associated with early onset of puberty in female mice and a tendency toward early puberty in male mice.(A) Relative Mkrn3 mRNA levels in the ARC of Mkrn3WT and Mkrn3KO females across PND10, PND15, PND20, and PND25 (n = 6 per genotype and age). Statistics were performed using 2-way ANOVA, followed by Tukey’s post hoc test. (B) Representative Western blot autoradiographic image of Mkrn3 protein and ß-actin in the MBH of Mkrn3WT and Mkrn3KO females at PND10. (C) Representative immunohistochemistry images of Mkrn3 protein in the POA and the arcuate nucleus (ARC) of Mkrn3WT and Mkrn3KO females at PND10. Scale bar = 100 µm. 3V, third ventricle. (D–G) Age at vaginal opening (D) and cumulative percentage of female mice exhibiting vaginal opening (E), and age at first estrus (F) and cumulative percentage of female mice with first estrus (G), in Mkrn3WT (n = 8) and Mkrn3KO (n = 10) females. (H) Age at preputial separation and (I) cumulative percentage of male mice exhibiting preputial separation in Mkrn3WT (n = 10) and Mkrn3KO (n = 11) males. Statistics were performed using unpaired 2-tailed t test. (J) Body weight of Mkrn3WT (n = 11) and Mkrn3KO (n = 15) male and Mkrn3WT (n = 8) and Mkrn3KO (n = 10) female mice measured weekly from weaning (PND21) to adulthood (PND84). Statistics were performed using 2-way ANOVA, followed by Tukey’s post hoc test. Data are presented as the mean ± SEM values. *P < 0.05, ***P < 0.0001 of Mkrn3KO compared with Mkrn3WT.

Fig 5: Mkrn3 deletion does not alter GnRH neuron morphology but increases spine density in the ARC.(A–C) Representative images of GnRH neurons classified into (A) unipolar, (B) bipolar, and (C) complex dendritic morphology. Scale bar = 50 µm. (D) Quantification of the percentage of GnRH neurons in the rPOA with unipolar, bipolar, and complex morphology in Mkrn3WT and Mkrn3KO females at PND15 (n = 5 per genotype). Statistics were performed using 2-way ANOVA, followed by Tukey’s post hoc test. Data are presented as median and distribution of the data and probability density (violin plot). (E) Representative images of Golgi-Cox–impregnated neurons in the ARC of Mkrn3WT and Mkrn3KO females at PND15. Scale bar = 5 µm. (F) Quantification of the number of dendritic spines/50 µm in ARC neurons of Mkrn3WT and Mkrn3KO females at PND15 (n = 5 per genotype), classified as thin, stubby, or mushroom according to their morphology. Statistical analysis was performed using unpaired 2-tailed t tests. Data are presented as median and distribution of the data and probability density (violin plot). *P < 0.05 compared with Mkrn3WT.