Discovery-based proteomics identifies N6-methyltransferases at sites of UNG2-seeded condensates.
(A) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-tagged cDNAs upon treatment with increasing concentrations of floxuridine at 64 hours post treatment.
(B) Quantification of experiment represented in A for percentage of cells with >5 mCherry foci.
Error bars, mean ± SEM; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance, ∗∗∗p ≤ 0.001, n = 3 biological replicates. Statistical tests performed within individual groups, EV or UNG2, respectively. (C) PONDR VSL2 plot of disorder for UNG2.
(D) Representative schematic of mutant UNG2 cDNA constructs expressed in UNG KO DLD-1 cells in E. IDR only cDNA lacks amino acids 93, ΔIDR cDNA lacks amino acids 1-92, and IDR-C cDNA moved amino acids 1-92 to the C-terminus. (E) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-Cry2-tagged cDNAs without or with stimulation of blue light for 60 seconds. While ΔIDR and EV images display cytoplasmic foci, these lack the distinct nuclear foci patterning observed for UNG2, IDR, and IDR-C constructs. (F) Schematic of proximity biotinylation of IDR interaction partners in cells. (G). Venn diagrams of factors identified by stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS) with 1>log2(L/H), reflecting a 4-fold enrichment in UNG-IDR/Control, from two biological replicates. n.s., non-statistically significant; Flox, floxuridine; EV, empty vector; IDR, intrinsically disordered region