title: Peptides modeled on the RGG domain of AUF1/hnRNP-D regulate 3' UTR-dependent gene expression.
abstract: Messenger RNA binding proteins control post-transcriptional gene expression of targeted mRNAs. The RGG (arginine-glycine-glycine) domain of the AUF1/hnRNP-D mRNA binding protein is a regulatory region that is essential for protein function. The AUF1-RGG peptide, modeled on the RGG domain of AUF1, represses expression of the macrophage cytokine, VEGF. This report expands studies on the AUF1-RGG peptide and evaluates the role of post-translational modifications of the AUF1 protein. Results show that a minimal 31-amino acid AUF1-RGG peptide that lacks poly-glutamine and nuclear localization motifs retains suppressive activity on a VEGF-3'UTR reporter. Arginine residues in RGG motifs may be methylated with resulting changes in protein function. Mass spectroscopy analysis was performed on AUF1 expressed in RAW-264.7 cells. In resting cells, arginines in the first and second RGG motifs are monomethylated. Following activation with lipopolysaccharide, the arginines are dimethylated. To evaluate if the arginine residues are essential for AUF1-RGG activity, the methylatable arginines in the AUF1-3RGG peptide were mutated to lysine or alanine. The R→K and R→A mutants lack activity. We also demonstrate that PI3K/AKT inhibitors reduce VEGF gene expression. Although immunoscreening of AUF1 suggests that LPS and PI3K inhibitors alter the phosphorylation status of AUF1-p37, mass spectroscopy results show that the p37 AUF1 isoform is not phosphorylated with or without lipopolysaccharide stimulation. In summary, arginines in the RGG domain of AUF1 are methylated, and AUF1-RGG peptides may be novel reagents that reduce macrophage activation in inflammation.
authors.affiliation: Veterans Administration Research Service, White River Junction, VT 05009, USA.
authors.forename: Abigail
authors.initials: A
authors.lastname: Fellows
authors.forename: Bin
authors.initials: B
authors.lastname: Deng
authors.forename: Dale F
authors.initials: DF
authors.lastname: Mierke
authors.forename: R Brooks
authors.initials: RB
authors.lastname: Robey
authors.forename: Ralph C
authors.initials: RC
authors.lastname: Nichols
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000502:physiology*; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D019869:Phosphatidylinositol 3-Kinases; D042461:Vascular Endothelial Growth Factor A; C000630987:Hnrpd protein, mouse
keywords: AU-rich element; AUF1; AUF1/hnRNP-D mRNA binding protein; AURE; Arginine methylation; GLUT1; LPS; MW; NLS; NLS-PY; RGG; RGG domain; TNF; UTR; VEGF; arginine-glycine-glycine; glucose transporter-1; hnRNP-D; lipopolysaccharide; mRNA binding protein; molecular weight; nt; nuclear localization signal; nucleotide; proline-tyrosine NLS; tumor necrosis factor-α; untranslated region of mRNA; vascular endothelial growth factor