Abstract 2087 Characterizing 14-3-3 proteins as putative RNA Polymerase II binding proteins

The carboxy-terminal domain (CTD) of RNA Polymerase II (RNA Pol II) serves as a binding platform for the recruitment of proteins which regulate progression of RNA Pol II transcription. Tandem heptad repeats of the consensus sequence YSPTSPS make up the CTD of RNA Pol II. Five of the seven residues in the heptad repeat - Tyr1, Ser2, Thr4, Ser5, and Ser7 – contain a hydroxyl group that is subject to posttranslational modification (PTM) by phosphorylation. Previous literature has shown that Thr4 phosphorylation (pThr4), a modification that is not as well characterized as other RNA Pol II PTMs, appears during the elongation stage and is reduced just before the polyA site in yeast. Though literature suggests a role for pThr4 in progression of RNA Pol II elongation, the precise function of this PTM in RNA Pol II transcription remains ambiguous. In this study, we conducted a pulldown experiment with HEK293 nuclear lysate to identify pThr4-interacting proteins. Initial hits of interest were three isoforms of the 14-3-3 protein (isoforms epsilon, theta, and zeta). This family of proteins contains seven isoforms in humans with over 100 known binding partners. Each 14-3-3 monomer contains a basic region the coordinates binding of either a phosphorylated serine or threonine residue. Here we characterize the affinity of 14-3-3 proteins for various CTD peptides. We found that all three 14-3-3 isoforms specifically bind a CTD peptide phosphorylated at the Thr4 position, but not at the Ser2 or Ser5 positions. We show that mutation of key basic residues in the binding pocket of 14-3-3 epsilon disrupts binding to a pThr4 CTD peptide. We also characterized these putative pThr4 CTD binding proteins in cells, demonstrating that the 14-3-3 epsilon isoform immunoprecipitates with RNA Pol II in a Thr4-dependent manner. We thank the National Institutes of Health (R01GM125882 and R35GM148356 to YJZ) for supporting our research.