(A) Domain organization of HEXIM1. The positions of the 23 lysines in HEXIM1 are indicated by yellow bars in the C-terminal (CT) and N-terminal (NT) regions and the NLS. (Below) Alignment of the NLS sequences from HEXIM1, UBE2O, and a known UBE2O substrate, BAP1 (Mashtalir et al., 2014). UBE2O and several of its target proteins contain a consensus bipartite NLS architecture that shares similarities with the HEXIM1 NLS. Basic residues are labeled in blue while hydrophobic residues are labeled in pink. (B) In vivo ubiquitination of HEXIM1 lysine mutants. Combinations of STREP-tagged lysine-to-arginine mutants were generated in the HEXIM1 N-terminal region (NTΔK), NLS (NLSΔK), and C-terminal region (CTΔK). A complete lysine-free HEXIM1 is designated as ΔK. UBE2O-F was co-transfected where indicated, and ubiquitination was detected by anti-HA western after denaturing lysis and IP. Monoubiquitin and multi-monoubiquitin species are indicated. The NTΔK mutant had no effect on ubiquitination, suggesting that there are no acceptor sites in the N-terminal region. However, both the NLSΔK and CTΔK mutants displayed decreased ubiquitination, demonstrating the presence of acceptor lysines in these regions. Schematic illustrates that lysines in the NLS (pink connection between domains) and in the C-terminal region function as acceptor sites. See also Supplementary file 2. (C) Coverage of the HEXIM1 sequence by MS. Sequences not identified by MS are bolded and underlined. Shaded in red is the HEXIM1 bipartite NLS. Color coding reflects whether a lysine was identified with a ubiquitin di-glycine tryptic remnant fragment. Shown below is a protein gel of HEXIM1-S purified under denaturing conditions from HEK 293 T cells with the co-expression of UBE2O-F. Arrows indicate unmodified and monoubiquitinated HEXIM1 species. (D) In vivo ubiquitination of individual lysine residues in the HEXIM1 NLS re-introduced in the context of the lysine-free (ΔK) HEXIM1 background. Ubiquitination of single-lysine HEXIM1 proteins was detected by HA Western blot. (top) All lysines are numbered in the HEXIM1 NLS and those that restore ubiquitination are indicated. (E) In vivo ubiquitination of wild-type STREP-tagged HEXIM1 or mutants deficient in binding P-TEFb (202PYNT205 -> 202PDND205) or the 7SK snRNA (150–156 Ala). HEXIM1-S was co-transfected with UBE2O-F as indicated.