Peptides modeled on the RGG domain of AUF1/hnRNP-D regulate 3′ UTR-dependent gene expression

• Published in: International immunopharmacology Vol. 17; no. 1; pp. 132 - 141
• Main Authors: Fellows, Abigail, Deng, Bin, Mierke, Dale F., Robey, R. Brooks, Nichols, Ralph C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2013
• Subjects: Amino Acid Sequence; Animals; Arginine methylation; AUF1; AURE; Cell Line; Gene Expression Regulation - physiology; Heterogeneous-Nuclear Ribonucleoprotein D - genetics; Heterogeneous-Nuclear Ribonucleoprotein D - metabolism; hnRNP-D; Macrophages - metabolism; Mice; Molecular Sequence Data; mRNA binding protein; Phosphatidylinositol 3-Kinases - genetics; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Protein Structure, Tertiary - genetics; RGG domain; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; hnRNP-D; AUF1; GLUT1; nt; VEGF; MW; TNF; LPS; NLS-PY; Arginine methylation; UTR; mRNA binding protein; RGG; NLS; AURE; RGG domain; AUF1/hnRNP-D mRNA binding protein; nuclear localization signal; untranslated region of mRNA; arginine-glycine-glycine; nucleotide; molecular weight; proline-tyrosine NLS; AU-rich element; tumor necrosis factor-α; vascular endothelial growth factor; glucose transporter-1; lipopolysaccharide
• ISSN: 1567-5769; 1878-1705
• DOI: 10.1016/j.intimp.2013.05.014

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. •AUF1-RGG peptides are modeled on the RGG regulatory domain of AUF1.•AUF1-RGG peptides decrease expression of VEGF.•Arginines in RGG motifs are methylated and are essential for AUF1-RGG peptide activity in macrophages.•The PI3K/Akt signaling pathway affects 3′ UTR-dependent VEGF gene expression.