Fig. 3: The THUMP and MTase domains of THUMPD2 contribute to U6 association, and SAM-binding and TRMT112-interaction are necessary for m²G₇₂ installation.

a Scheme of THUMPD2 domain architecture (aa amino acid, MTase methyltransferase, NLS nuclear localization signal). Red lines indicate amino acids crosslinked to RNA in (b; confidence scores >0.15). b Cells over-expressing THUMPD2-His₆-2xFLAG were crosslinked (UV and PAR; n = 1 each) and protein-RNA crosslinked peptides in immunoprecipitations eluates were identified by mass spectrometry. c Model of U6 (light purple; G₇₂ – sticks, N2 atom – blue sphere) bound to the AlphaFold model of THUMPD2-TRMT112 (TRMT112 – raspberry; THUMPD2 - THUMP domain – cyan, MTase domain – light green, NLS – dark blue, ⁴⁸³VSLGKT⁴⁸⁸ loop – magenta, SAM – orange (methyl group – sphere)). The crosslinked amino acids from (a, b) are identified in red. d Profile of CRAC reads mapping to each nucleotide of U6 is shown for one representative experiment. Normalized read counts are depicted as reads per million mapped reads (RPM). e Magnified view of amino acid substitutions anticipated to disrupt SAM binding (D329A) and TRMT112 interaction (E352R). f Predicted bipartite NLS in THUMPD2. g, h HEK293 cells over-expressing GFP-ΔNLS-THUMPD2 (g) or truncated versions of THUMPD2 N-terminally tagged with GFP (h) were used in fluorescence microscopy n = 3 experiments. Nuclear material was stained using DAPI (scale bars = 10 µm). i Lysates from cells over-expressing His₆-2xFLAG, THUMPD2-His₆-2xFLAG WT and variants (NTD and CTD) were used in n = 4 immunoprecipitation experiments. Proteins and RNAs in input and eluate samples were analyzed by western and northern blotting, respectively. j Schematic view of base-pairing of the m²G-sensitive DNAzyme with U6 snRNA to detect methylation of G₇₂. k U6 ISL RNA oligonucleotides (nucleotides 55–80) with G₇₂ or m²G₇₂ were incubated with the DNAzyme and the fraction of cleaved RNA at the indicated times was determined. Data from n = 3 independent experiments are shown as mean ± standard deviation. l Proteins from WT and THUMPD2 KO cells, and THUMPD2 KO cells expressing the indicated constructs were analyzed by western blotting. Tubulin served as a loading control, and antibodies against endogenous THUMPD2 and the FLAG tag were used in the left and right panels, respectively. Total RNAs were left untreated (-DNAzyme) or subjected to DNAzyme-mediated cleavage ( + DNAzyme). U6 and U4 snRNAs were detected by northern blotting. m Quantification of full-length U6 relative to the cleaved fragment after DNAzyme treatment. Ratios were normalized to WT samples. Data from n = 3 independent experiments (l) shown as mean ± standard deviation. Source data are provided as a Source Data file.