The post-translational methylation of arginine in the glycine arginine rich region of CHO nucleoin

Nucleolin is a nucleolar protein important for ribosome biogenesis. Nucleolin contains a conserved glycine arginine rich (GAR) domain near its carboxy terminus. GAR domains are defined by repeating tri-peptide, (arginine -glycine-glycine (RGG)) motifs. The arginine in the RGG motif is post-translationally modified by dimethylation on one of the two guanido nitrogens. Although arginine methylation was identified over 30 years ago, the function of this modification remains unknown. The GAR domain of nucleolin is important for nucleic acid binding and for nucleolar localization of nucleolin. This dissertation describes investigates possible in vivo interactions between nucleolin and other nucleolar proteins involved in ribosome biogenesis. This dissertation also addresses the possible roles that arginine dimethylation may play in the function of the GAR domain. A GAR truncated hamster nucleolin (DGAR) localizes to the nucleoplasm and not to the nucleoli of CHO cells. This version of nucleolin was subsequently used to probe the in vivo interactions of nucleolin with other non-ribosomal nucleolar proteins. In support of previous work, DGAR caused endogenous B23 to redistribute to the nucleoplasm, suggesting an in vivo interaction. Endogenous Nopp140 shows no redistribution in the presence of DGAR. The effect of DGAR on the nucleolar protein, fibrillarin, remains unclear. A redistribution of exogenously expressed wild type nucleolin from nucleoli to the nucleoplasm is observed when CHO cells are treated with the methyltransferase inhibitors 5-methyl 5¢ deoxythioadenosine (MTA) and 3-deaza-adenosine (DAA). This redistribution of nucleolin is reversible and independent of protein synthesis. The arginines in the RGG motifs of the GAR domain of hamster nucleolin were changed to lysine, and the fully substituted protein was not a substrate for the RGG specific protein arginine methyltransferase, Hmt1p. The lysine substituted protein bound nucleic acids and behaved in vivo in a manner indistinguishable from wild type nucleolin. These results indicate that methylation is not necessary for in vitro nucleic acid binding or for in vivo localization of nucleolin, and that the redistribution of nucleolin observed following MTA treatment is likely due to inhibiting the methylation of another nucleolar substrate