Schematic representation of the Set1 and subunit major interactors identified in the systematic yeast two-hybrid screens.

A) Schematic representation of Set1 FL and Set1 fragments 1-754 and 754-1081. B) Set1 FL, 1-754 and 754-1081 major Y2H interactors). C) Interacting regions of Set1 with Swd2, Shg1 and Spp1. D) Swd2, Spp1, Shg1 major Y2H interactors. E) Swd1, Swd3, Bre2, Sdc1 major Y2H interactors. The term “interactor” is used to mean a high confidence two-hybrid interaction, with the limitations that this entails. The color reflects the Predicted Biological Score (see METHODS). Red, highest confidence; Blue, high confidence; Green, good confidence.

Set1 1-754 interacts with RGG proteins and the importin Kap104.

A) RGG proteins and import/export proteins interacting with Set1 1-754, Set1 754-1081 and Spp1, Shg1, and Sdc1. B) Set1 interacting domain (SID) (blue) within Kap104. Heat like repeat 9 and 10 are represented in purple C) PY-NLS in the N-terminal region of Set1. D) AlphaFold modelling of a seven subunit Set1C (Set1-Bre2-Sdc1(x2)-Swd1-Swd3-Spp1) and Kap104. A representative model is shown using the following colour code: Kap104 SID 359-621 (white, the remaining Kap104 residues are hidden. Set1 N-term 1-754 (yellow), Set1 C-term 755-1079 (red); Set1 PY-NLS 41-90 (blue); Swd1 (green); Swd3 (cyan); Bre2 (raspberry); Sdc1 (subunit 1, grey); Sdc1 (subunit 2, chocolate); Spp1 (violet).

The SET1C Y2H interactome identifies proteins involved in RNA biogenesis.

All Y2H interactors are described in Table S2. The processes linked to RNA metabolism in which the different interactors are involved are shown in the figure. The green lines linking Prp22 to Set1FL/Set1 754-1080 and Prp8 to Set1 FL and Spp1 indicate interactions with a high degree of confidence.

Set1 is co-precipitated with Prp22 in vivo.

A) Co-immunoprecipitation experiments were performed in W303 expressing chromosomally encoded Myc-Set1 (Dehe et al. 2006) and Prp22AID-FLAG (Mendoza-Ochoa et al. 2019. Prp22-AID-6FLAG was pulled down using a FLAG antibody. IgG was used as a negative control. Input, non-bound and immunoprecipitation (IP) samples were loaded. The Western blot was probed either with an antibody against the MYC epitope tag (Left) or against the FLAG epitope tag (Right). Myc-Set1 and Prp22AID-FLAG are indicated by * and °, respectively. B and C) The same experiment was carried out in the absence or presence of RNase. A strain with 9MYC-Set1 and no flag-tagged Prp22 was used as a negative control. The Western blots were probed with anti-MYC and anti-FLAG respectively.

Set1 is SUMOylated.

6His-SUMO–conjugated proteins were purified from cells transformed (+) or not transformed (−) with a plasmid encoding 6His-SUMO under control of the CUP1 promoter. Cell lysates (Input) and Ni-purified material (Elutes) were analyzed by Western blotting with an anti-MYC antibody (A) or and anti-GAL4 antibody (B-D). Analysis of 6His-SUMO-conjugated forms of (A) genomically MYC-tagged Set1 or (B) GB-Set1 transformed cells was performed (left panels), in both the cases SUMO expression and efficiency of purification were controlled using an anti-SUMO antibody (right panels). C) SUMOylation analysis of Set1 fragment F3+F4 (aa. 351-956) WT and the K769R mutant. D) SUMOylation analysis of Set1 fragment F5 (aa. 956-1080) WT, single mutants K1055R and K1060R, and the double mutant K1055R/K1060R mutant. Unmodified MYC-Set1 and GAL4-Set1 in both the (-) and (+) His-SUMO eluates are most likely due to the stickiness of unmodified Set1 to the beads.

SET1C interacts in vitro with Snf2C-AT-hook.

A) A schematic diagram depicting the domains of Snf2 and the Snf2 fragments used in this study, along with the SDS-PAGE/Coomassie staining of the purified GST-tagged Snf2 fragments. B and C) GST pull-down assay using purified GST-tagged Snf2 fragments. The purified SET1C (B) or SET1C-C762 complex (C) was mixed with GST-tagged Snf2 fragments, followed by GST pull-down, and the bound proteins were analyzed by immunoblotting. D) A schematic diagram illustrating Snf2 fragments with a more detailed breakdown of the AT-hook domain, along with the SDS-PAGE/Coomassie Blue staining of the purified Snf2 fragments. The lysines present in the AT-hook are represented by the letter K. E) GST pull-down assay using purified GST-tagged Snf2 fragments and SET1C.

Snf2 is methylated in tandem AT-hook domain by reconstituted Set1C.

A) In vitro methyltransferase assay using purified SET1C and Snf2 fragments. 3H-SAM was used as a methyl-donor and methylated proteins were detected by autoradiography. The band marked with a red star is a degradation product of Snf2C. B) In vitro methyltransferase assay using SET1C and two Snf2-AT-hook fragments with two different tags. C) Schematic diagram of N-terminal truncated SET1 complexes (left) and in vitro methyltransferase assay with GST-Snf2-AT-hook and truncated SET1 complexes. D) Schematic diagram showing the positions of all lysines in Snf2-AT-hook and the further cleaved fragments of Snf2-AT-hook. The red box indicates the two lysines that are acetylated by Gcn5. E) Coomassie staining of purified Snf2 fragments (lower) and an in vitro methyltransferase assay using these fragments with SET1C (upper). F) In vitro methyltransferase assay by SET1C when each of the four lysines in the C-terminal region of the Snf2-AT-hook is substituted with arginine or when both lysines known to be acetylated by Gcn5 are substituted. G) A schematic diagram showing the position of the RG-repeat region and the design of Snf2-AT-hook with RG-repeat truncation. H and I) GST pull-down assay (H) and in vitro methyltransferase assay (I) using purified SET1C and GST-Snf2-AT-hook with or without RG-repeats.

The arginines in the RG-repeat of Snf2 are methylated by reconstituted SET1C.

A) A diagram showing the WT Snf2-B3 fragment and the Mut Snf2-B3 with all four lysines substituted with alanine. B) Coomassie staining of purified WT and Mut Snf2-B3. C) Mass-spectrometry experiment design to identify the methylation sites of Snf2-B3. D) Coomassie staining of Mut Snf2-B3 after methylation reaction and an additional purification step using Ni-NTA. The band corresponding to Mut Snf2-B3 (∼18 KDa) was excised and used for mass spectrometry analysis. The ∼37 KDa band observed in lanes 5–8 appears to be a SET1C subunit that binds non-specifically to Ni-NTA, likely SWD2 based on its size. E) Mass spectrometry analysis result of Snf2-B3 methylation sites revealed that multiple arginines in the RG-repeat were methylated. Amino acid sequence of Snf2 highlighting the arginines methylated by SET1. The sequence of the B3 fragment is shown in bold, and the arginines methylated by SET1 are marked in red. Methylation and demethylation are denoted as M and DM, respectively.

The arginines of the motif ARTSTRGR within the AT hook are methylated in vivo in a Set1-dependent way.

A) Set1 interacts in vivo with Snf2 and Snf2-ΔRG. Myc-tagged Snf2 and Snf2ΔRG were immunoprecipitated with 9E10 Myc antibodies (see Methods) and revealed with either 9E10 (Upper panel) or Set1 antibodies (lower panel). B) Snf2C complex was purified from WT and set1Δ strains, separated on a 4-12% Bis-Tris Gel and Silver Stained (Left); the presence of Snf2-GFP is detected by Western-blotting with anti-GFP antibody (Right). The area corresponding to Snf2-GFP was excised from the gel and used for mass spectrometry analysis. Peptides flanking the RG repeats (1485-1549) with their PTM are shown in Fig. S15. C) Panel C show a focus on the amino sequence flanking the RG repeats. The positions of residues from D1485 to V1549 are indicated on the figure. Peptides identified after digestion of Snf2-GFP are indicated in color with their identified PTM indicated by the color code shown in the top of the panel. The small numbers above the amino acids indicate the probability of localisation according to the MS2 peaks. It should be noted that for wild-type K1488 and R1490, a peptide with dimethylation is detected, but no discriminating MS2 peak allows us to conclude whether K or R are dimethylated. This is also the case for dimethylation on R1528 and R1535. The results presented represent the observed PTMs from two independent experiments, each containing 3 replicates (Table S6).