Subcellular localization of the RNA‐binding protein PTBP1 is regulated by post‐translational modifications in the RRM2 domain

Abstract

Alternative splicing is an important mechanism that increases protein diversity and contributes to the modulation of gene expression. Polypyrimidine tract binding protein 1 (PTBP1) regulates alternative splicing of pre‐mRNA and polyadenylation in the nucleus, and functions in mRNA localization, stability and translation in the cytoplasm. To understand the regulation of the nucleocytoplasmic shuttling of PTBP1, a structure‐function analysis has been performed. Previous studies indicated that nuclear export is promoted by both the classic nuclear export sequence and the RRM2 domain, and recently we reported that several lysine residues in the RRM2 domain are post‐translationally acetylated (Pina et al. 2018). Therefore, to examine the role of lysine acetylation in nuclear export, human osteosarcoma MG63 cell were stably transfected with either wild type FLAG‐PTBP1 or one of four FLAG‐PTBP1 constructs carrying individual lysine‐to‐alanine mutations (K212A, K218A, K259A, or K266A). Wildtype PTBP1 was sometimes cytoplasmic, but remained predominantly nuclear in immunofluorescence studies, in agreement with previous reports (Kamath et al. 2001, Li et al. 2002, Xie et al. 2003). Mutants K266A and K212A were predominantly cytoplasmic. Since the loss of these lysine residues (that we discovered are acetylated under splicing conditions) appeared to promote nuclear export, our working hypothesis is that acetylation of these residues inhibits nuclear export. The change in localization of these mutant proteins is not activity‐based, since neither of the mutations affected the ability of PTBP1 to regulate splicing in a cell‐based assay. The other two mutants, FLAG‐PTBP1 K259A and K218A, were more exclusively nuclear than the wild type protein, suggesting that these mutations might also affect nuclear export. These two mutations do not affect PTBP1 activity in a splicing assay. Currently these cell lines are being tested in a classic heterokaryon nuclear export assay in which the human MG63 cell lines are fused to mouse C2C12 cells and then, using an immunofluorescence assay, PTBP1 movement between nuclei is detected. To our knowledge, this is the first study to investigate the role of RRM2 in PTBP1 nuclear export.Support or Funding InformationThis work was supported in part by NIH SC3 Award ( 1SC3GM132036‐01) to N.K. Additional funding was provided by the Department of Biological Science research funds and BIOL 418L course funding (A. Miyamoto, A. Medina), CIRM Bridges to Stem Cell Research (D. Peralta), Project RAISE (L. Gonzalez), and Research Careers Preparatory Program (E. Caballero).