Fig 2: SPOC domain proteins regulate gene expression and m6A levels.a, b, d, e, g–i MA plots showing RNA-seq log2 fold change (KO/WT or ?SPOC/WT) versus log10 mean expression in WT for a PKF3 KO, b PHF3 ?SPOC, d DIDO KO, e DIDO ?SPOC, g RBM15 KO, h RBM15 ?SPOC, and i SHARP ?SPOC. Red and blue dots indicate upregulated and downregulated genes, respectively with fold-change > 2, p < 0.05. Statistical analysis was performed using the Wald test as implemented in DESeq272. Drosophila S2 cells were used for spike-in normalization. c, f, j Venn diagram showing overlaps between c upregulated genes in PHF3 KO and PHF3 ?SPOC, f downregulated genes in DIDO KO and DIDO ?SPOC, j downregulated genes in SHARP ?SPOC, RBM15, and RBM15 ?SPOC. k m6A levels are decreased upon impairment of RBM15. Mass spectrometry analysis of single nucleosides derived from mRNA isolated from the indicated cell lines. Data are presented as mean ± standard deviation of three replicates, and individual data points are indicated as black dots. One-tailed, two-sample equal variance t-test was used to determine p-values. Source data are provided as a Source Data file.

Fig 3: SPOC bridges transcription with co-and post-transcriptional processes.SHARP and RBM15 interact with RNA and writers and readers of m6A RNA modification, while PHF3 and DIDO interact with regulators of transcription and co-transcriptional processing. SPOC protein interactions, in turn, regulate downstream processes like translation and mRNA decay.

Fig 4: Acidic residues determine SHARP SPOC binding affinity.a Fluorescence anisotropy binding curves of SHARP SPOC with NCoR peptides. Fluorescence anisotropy is plotted as a function of protein concentration. Data points and error bars represent the mean ± standard deviation from three independent experiments. Source data are provided as a Source Data file. b Crystal structure of SHARP SPOC in complex with 1×S5P CTD peptide (7Z1K). Electrostatic surface potential was calculated using the Coulombic Surface Coloring tool in UCSF Chimera and is depicted ranging from -10 (red) to +10 (blue) kcal/(mol*e). c Structural comparison of SHARP SPOC binding to 1×S5P CTD and SMRT (2RT5). d Interactions between SHARP SPOC and 1×S5P CTD. Hydrogen bonds are indicated with dashed lines. The blue circle indicates a conserved basic surface patch. e Interactions between SHARP SPOC and phosphorylated SMRT (2RT5). Hydrogen bonds are indicated with dashed lines. The blue circle indicates the conserved basic surface patch, red circle indicates additional electrostatic coordination of R3548 of SPOC by D2523 and E2525 of SMRT, which is not possible with CTD. f Interactions between PHF3 SPOC and 2×S2P CTD (6Q2V). Hydrogen bonds are indicated with dashed lines. Blue circles indicate conserved basic surface patches. g HADDOCK scores of modeled interactions between SHARP SPOC or RBM15 SPOC and SMRT, CTD, or FMR1 peptides. Peptides used for modeling were either unphosphorylated (white bars) or phosphorylated at the indicated residues (SHARP SPOC: orange bars, RBM15 SPOC: gray bars). Bars correspond to the HADDOCK score ± SD calculated by HADDOCK. Source data are provided as a Source Data file.

Fig 5: Interactome of SPOC proteins.a Western Blot analysis after FLAG-co-IP of FLAG-tagged SPOC domains. b–e Western Blot analysis after FLAG-co-IP of FLAG-tagged full-length and ?SPOC proteins. The experiments in a–e were performed once. f Fluorescence anisotropy binding curves of RBM15 SPOC with WTAP peptides. Fluorescence anisotropy is plotted as a function of protein concentration. Data points and error bars show mean anisotropy ± standard deviation from three independent experiments. g Structural model of the interaction between SHARP SPOC and an 8-mer FMR peptide phosphorylated at S511 generated in PyMOL and refined using the HADDOCK2.2 webserver28,29. Repulsion between SHARP R3566 and FMR R507 may lead to reduced binding affinity compared to SMRT. h Fluorescence anisotropy binding curves of SHARP SPOC with FMR1 peptides. Fluorescence anisotropy is plotted as a function of protein concentration. Data points and error bars show mean anisotropy ± standard deviation from three independent experiments. Source data are provided as a Source Data file.