Dr. Ming F. Tam 譚鳴輝博士

Research

Cellular Responses to Protein Arginine Methylation

The main interest of our laboratory is on proteins with arginine side chain methylation and the subsequent cellular responses. There are at least three types of protein arginine methyltransferases (PRMTs). The type I enzymes mediate the formation of ω-N^G-monomethylarginine (ωMMA) and asymmetric ω-N^G-N^G-dimethylarginine (aDMA). The type II enzymes have the symmetric ω-N^G-N^G'-dimethylarginine (sDMA) in addition to the ωMMA as products. The δ-guanidino nitrogen of the arginine side chain can also be methylated with a type IV PRMT from the yeast. Asymmetrical dimethylation of arginines has been shown to influence mRNA nuclear export, protein-protein interactions and transcription. Symmetrical dimethylarginine methylation is required for the localization of SMN (survival of motor neurons) in Cajal bodies and pre-mRNA splicing. Proteins with monomethyl arginine have been observed in tumor cells during apoptosis. The cellular functions of proteins with monomethylarginine are at present unknown.

Our laboratory is using yeast as a model system to study the cellular responses to protein arginine methylation. We have identified Stm1p as a substrate of Hmt1p which is a type I PRMT. Stm1p binds quadruplex DNA but resides primarily on ribosome. Stm1p is methylated at Arg237. Stm1p with Arg237 to lysine substitution is more effective than the wild type protein as multicopy suppressor of the staurosporine- and temperature-sensitive phenotypes of a pop2 yeast strain. Experiments are underway to determine whether Stm1p methylation affects ribosome assembly and/or chromosome binding.

Sbp1p is another substrate of Hmt1p. Sbp1p is a nucleolar single-strand nucleic acid binding protein that we found associated with the ribosome. Sbp1p interacts with numerous proteins but only the methylated Sbp1p can bind Nop13p, Mas1p and Rpl16Ap. The results implicate the involvement of methylated Sbp1p in rRNA processing.

Ribosomal protein Rpl12p is δ-monomethylated at Arg67 by RMT2. Yeast strain with RPL12A and RPL12B double deletion exhibits slow growth and slow translation. Cells transformed with plasmids encoding the wild type Rpl12p or its Arg67 to lysine mutant have different growth rate. We are testing whether mRNAs are preferentially translated on the polysomes of these strains.

Detection of methylated amino acids with mass spectrometry. Methylated Sbp1p (panel A) and Sbp1p (panel B) were isolated on gels then subjected to acid hydrolysis. Resulting amino acids were detected on a time-of-flight (TOF) mass spectrometer with matrix-assisted-laser-desorption ionization (MALDI). The dimethylarginine (DMA, m/z 203) can be clearly detected in the methylated Sbp1p sample.