Abstract
Ubiquitin-specific proteases (USPs) USP15 and USP4 belong to a subset of USPs featuring an N-terminal tandem domain in USP (DUSP) and ubiquitin-like (UBL) domain. Squamous cell carcinoma antigen recognized by T-cell 3 (SART3), a spliceosome recycling factor, binds to the DUSP-UBL domain of USP15 and USP4, recruiting them to the nucleus from the cytosol to control deubiquitination of histone H2B and spliceosomal proteins, respectively. To provide structural insight, we solved crystal structures of SART3 in the apo-form and in complex with the DUSP-UBL domain of USP15 at 2.0 and 3.0 Å, respectively. Structural analysis reveals SART3 contains 12 half-a-tetratricopeptide (HAT) repeats, organized into two subdomains, HAT-N and HAT-C. SART3 dimerizes through the concave surface of HAT-C, whereas the HAT-C convex surface binds USP15 in a novel bipartite mode. Isothermal titration calorimetry measurements and mutagenesis analysis confirmed key residues of USP15 involved in the interaction and indicated USP15 binds 20-fold stronger than USP4.
Highlights
Ubiquitination plays an important role in almost every biological process, including protein homeostasis, DNA damage response, gene transcription, protein trafficking, and RNA splicing
USP4, USP11, and USP15 are a small set of closely related Ubiquitin-specific proteases (USPs), sharing a similar domain architecture as follows: a domain in USP (DUSP), followed by a ubiquitin-like (UBL) and a large catalytic domain, bifurcated by a second UBL domain and disordered region (Fig. 1A)
USP4 is recruited by U4/U6 small nuclear RNA recycling factor SART3 to remove the Lys-63polyubiquitin chain from pre-mRNA processing factor 3 (Prp3), and it controls the assembly of the spliceosome at distinct stages of the splicing process (4)
Summary
Ubiquitination plays an important role in almost every biological process, including protein homeostasis, DNA damage response, gene transcription, protein trafficking, and RNA splicing. SART3 plays multiple roles in mRNA splicing, viral and host gene transcription, as well as stem cell survival, proliferation, and differentiation. It is a potential antigen for cancer immunotherapy (7–9). SART3 is a large protein of 110 kDa and contains multiple half-a-tetratricopeptide (HAT) repeats followed by a bipartite nuclear localization sequence (NLS), two RNA recognition motifs, and a C-terminal conserved LSm-interacting motif (Fig. 1A). The central HAT repeat region of SART3 mediates direct interactions with the DUSP-UBL domains of USP4 (4) and USP15 (5) but not of USP11. We report the crystal structure of the first eight HAT repeats of SART3 and SART3 in complex with the DUSP-UBL domain of USP15. Binding affinity measurements and mutagenesis analysis confirmed the interaction between
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