Extended Data Fig. 2: PPIA functional validation experiments.
a, Interaction between PPIA and PABPC1 in haematopoietic cells. Co-IP followed by MS/MS identifies PABPC1 as an interactor of PPIA in the human haematopoietic cell lines THP1 (acute monocytic leukemia, AML) and NB4 (acute promyelocytic leukemia, APML). Cells were transduced with 3XF-tagged PPIA (3XF-WT-PPIA or 3XF-Mutant(G104A)-PPIA, respectively) and IP was performed with an anti-3XFLAG antibody. The arrow indicates the band for PABPC1 protein; (representative of two independent experiments shown). b, Identification coverage of PABPC1. Co-immunoprecipitated bands were excised after SDS-PAGE (Fig. 2b), trypsin digested, and identified by MS/MS. Coverage for PABPC1 extends to 26.4% of the protein, including the N-terminal RNA-binding domain, the C-terminal domain interacting with cap-binding proteins, and the unstructured linker region; (representative of two independent experiments shown). c, List of nucleotide-binding PPIA client proteins. Proteins listed by their official gene names. Nucleotide binding was determined based on UniProt classifications. d, Efficiency of PPIA knockdown in 293T cells and HeLa cells. Cells were stably transduced with pLKO.1-TRC control, TRC PPIA Kd1, or TRC PPIA Kd2 lentiviral vectors, respectively. Then, cell lysates were prepared and loaded onto a SDS-PAGE in order to measure PPIA protein expression by westernblot using a rabbit polyclonal anti-PPIA antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control for protein normalization; (representative of three independent experiments shown). e, Presence of intrinsically disordered regions correlates with a slower translation rate. Reanalysis of data from Schwanhäusser et al.35 showing the inverse correlation between protein translation speed and percentage of intrinsically disordered regions36 in the whole proteome. Proteins with more intrinsically disordered regions translate at a slower rate than structured proteins. Details on the calculation of protein synthesis rates have been described by the original authors35.
