Fig 1: INTS13 associates with the INTS4/9/11 heterotrimer.a Yeast two hybrid screening for interactors of INTS13. Haploid strains of yeast expressing each individual Integrator subunit fused to the Gal4 activating domain (AD) were mated with strains expressing INTS13 fused to the DNA binding domain (BD). The first and third panels show mated yeast grown on selective media as a positive control (media lacking leucine, tryptophan, and uracil). The remaining panels are of yeast plated on restrictive media additionally lacking histidine to test for protein-protein interactions and with 3AT (3-amino-1,2,4-triazole) to test the strength of the interaction. No interaction was observed between INTS13 and any other individual subunit (second panel). When INTS9 and INTS11 were expressed in trans with INTS13, there was a positive interaction with INTS4 (fourth panel). b Point mutants of INTS4, INTS9, and INTS11 known to disrupt their interactions were used in yeast two hybrid to demonstrate that the formation of the INTS4/9/11 heterotrimer is necessary for INTS13 association. General locations of the mutations are indicated with arrows on the schematic. INTS13 is fused to the activating domain in the rest of the yeast two hybrid assays shown in this study. c SDS PAGE gel of fractions from gel filtration column of co-expressed full-length INTS4, INTS9, INTS11 and INTS13. The sample was first purified by nickel affinity chromatography. INTS4 carried an N-terminal His-tag, while the other three proteins were untagged. The positions of the fractions are indicated at the top of the gel and molecular weight markers shown are in kilodaltons. Multiple purifications were conducted (> 3), and the gel shown is representative of the results attained. d Consecutive 200-amino acid truncations of INTS13 were tested for interaction with INTS4/9/11. In modified yeast two-hybrid assays, the only positive interactions seen are full length INTS13 and the last ~200 residues, 501-706. The patient mutations are marked on the schematic and occur in this region. Loss of interaction with INTS4/9/11 was observed using the mutant versions of INTS13. Source data are provided as a Source Data file.