Fig 2: YTHDF2 protein levels in the nucleus change in response to heat shock, and MAGEL2 expression blunts this response.A, HEK293-MAGEL2 cells that were either induced with tetracycline to express MAGEL2, or uninduced, were placed in a 42 °C water bath for 1 h. Cells were harvested at different time points, before heat shock (Pre-HS), immediately following heat shock (Post-HS) or at 1, 2, and 4 h following removal of the samples from the water bath. Proteins from cellular fractions (whole cell, WC, or nuclear fraction, Nuc, or cytoplasmic fraction, Cyto) were immunoblotted to examine the abundance of endogenous YTHDF2. Equal amounts of protein were loaded. A representative trial is shown. B, the change in the proportion of YTHDF2 in the nucleus relative to preheat shock in induced (MAGEL2-expressing) and uninduced cells was plotted over time for triplicate samples, normalized to the amount of YTHDF2 in the nuclear fraction a pre-heat shock (uninduced, black and induced, red, mean ± standard deviation, individual triplicate values are also shown). The relative amount of YTHDF2 protein in the nucleus increases after heat shock in uninduced (black, no MAGEL2 expression) HEK293-MAGEL2 cells (repeated measures ANOVA, ∗p < 0.01, n = 3). There was no change in relative levels of nuclear endogenous YTHDF2 levels after heat shock in induced, MAGEL2-expressing cells (red circles, repeated measures ANOVA, ∗p > 0.05, n = 3).

Fig 3: Human fibroblasts from individuals with PWS have less YTHDF2 protein than control fibroblasts, and endogenous USP7 and YTHDF2 proteins coimmunoprecipitate with MAGEL2.A, endogenous YTHDF2 protein levels were measured by immunoblotting lysates from cultured immortalized fibroblasts. C12 and C14 are fibroblast lines from control individuals, whereas P16 and P21 are fibroblast cell lines from individuals with PWS lacking MAGEL2 expression. Right, analysis of replicate blots of YTHDF2 levels in control and PWS fibroblasts, with equal amounts of total protein loaded per lane (mean ± standard error of the mean, n = 8 per genotype, ∗p = 0.03 by Student t test). B, quadruplicate samples of MAGEL2-HEK cells were induced (I) or uninduced (U) to express full-length MAGEL2 (M). Endogenous YTHDF2 and endogenous USP7 coimmunoprecipitated with FLAG-MAGEL2 using anti-FLAG M2 gel. 10% of each cell lysate was immunoblotted to confirm the presence of all proteins in the input lysates.

Fig 4: Protein–protein interaction analysis of proteins in proximity to MAGEL2 as detected by BioID-MS.A, the 34 proteins identified in at least two out of three replicate BioID-MS experiments with MAGEL2 and not eliminated as background contaminating proteins were analyzed. Interactions among MAGEL2-proximal proteins were identified, revealing eight clusters of proteins. Proteins outlined with thicker lines were found in three out of three replicates. B, interactions among proteins proximal to full-length MAGEL2 but not CtermMAGEL2 and deduced to be proximal to the N-terminal, intrinsically disordered portion of MAGEL2. Proteins found in all three replicates of the BioID for full-length MAGEL2 and none of the replicates for C-terminal MAGEL2 are outlined with thicker lines.