Fig. 6
PINT87aa directly interacts with the PAF1 complex and inhibits mRNA transcriptional elongation. a Left, immunoprecipitation was performed using an anti-Flag antibody in PINT87aa-3XFlag or empty vector transfected 293T cells. Western blotting using an anti-Flag antibody confirmed PINT87aa overexpression. The precipitates were subjected to LC-MS/MS to identify potential PINT87aa-interacting proteins. Right, PAF1 complex-related proteins were identified in PINT87aa precipitates. b Immunoprecipitation was performed in PINT87aa-3XFlag-transfected cells or control cells. Western blotting was performed using an anti-PAF1 antibody. c Upper, PINT87aa conformations were modeled with PEP-FOLD and docked to PAF1 based on the ATTRACT2 force field using the PEP-SiteFinder pipeline. PINT87aa was split into three segments: 1–36 aa, 27–62 aa, and 53–87 aa. The top ten peptides in complex with PAF1 were visualized with different colors using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.), while the protein–peptide interface residues (within 5 Å for each peptide) were labeled. Lower, the direct mutual interactions of PINT87aa with different domains of HA-tagged PAF1 were tested using purified proteins (Flag-tagged PINT87aa and HA-tagged PAF1). d IF was performed to determine PINT87aa-PAF1 colocalization in PINT87aa-GFP transfected 293T cells or hNSC. Scale bar, 20 μM. e The expression of PAF1 downstream genes was determined by performing Western blotting and q-PCR in PINT87aa- or circPINTexon2-overexpressed 456 and 4121 BTIC and their respective controls. Data are resented as mean ± s.e.m. from three independent experiments. **P < 0.01, ns, P > 0.05, determined by two-tailed Student’s t-tests
