Fig. 6: PHF3 negatively regulates mRNA stability via the SPOC domain.
From: PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC
a Density distribution of the differences in log2 fold changes PHF3 KO/WT or PHF3 ΔSPOC/WT between RNA-seq and PRO-seq data. b Comparison of mRNA half-lives for 757 genes calculated from T-C conversion rates as determined by SLAM-seq in PHF3 WT, KO, and ΔSPOC cells (n = 6). Median Spearman correlation coefficient of conversion rates for replicate samples belonging to the same group (same genotype and timepoint) was 0.75 (see Supplementary Fig. 12c). The difference between the distributions is statistically significant based on the one-sided Wilcoxon test [P(KO – WT) = 1.34 × 10−11, P(ΔSPOC – WT) = 2.28 × 10−11]. Statistics are indicated in detail in Supplementary Data 7. c, Scatter plot showing correlation between half-lives in PHF3 ΔSPOC and PHF3 KO. Spearman’s correlation coefficient is indicated. d, e Conversion rates determined from targeted SLAM-seq analysis of d, INA mRNA and e, NAT10 mRNA as a control labeled with s4U for 12 h followed by pulse chase for 6 h and 12 h. Robust linear models were fit on the linearized form of the exponential decay equation. Y-axis shows the log2 conversion rate, shifted by the median conversion rate at t = 0 h. For INA: t1/2 = 3.3 h (WT), 7.1 h (ΔSPOC), 19.5 h (PHF3 KO). For NAT10: t1/2 = 3.3 h (WT), 4.3 h (ΔSPOC), 5.0 h (PHF3 KO). f, g Relationship between RNA-seq fold change and half-life fold change for f, PHF3 KO vs WT or g, PHF3 ΔSPOC vs WT. The majority of differentially regulated genes cluster in the top right quadrant that corresponds to mRNAs with increased steady-state levels and half-lives. h, i Relationship between t10 elongation rate (10 min after DRB washout) fold change and half-life fold change for h, PHF3 KO vs WT or i, PHF3 ΔSPOC vs WT.
