Fig. 1.

Structural and functional conservation of PRMT5. (A) A schematic representation of domain structures of C. elegans and human PRMT5, and a representative type-I arginine methylase, PRMT1 of rat. The lengths of the boxes are approximately drawn in scale with the protein lengths, and the residue numbers at domain boundaries are labeled. Areas filled in tan, cyan, green, and yellow represent TIM-barrel, Rossmann-fold, β-barrel, and oligomerization domains, respectively. Levels of amino acid identity and similarity of the individual domains between C. elegans and human PRMT5s, and that between human PRMT5 and rat PRMT1 are shown. (B) Sequence alignment. The full-length sequences of C. elegans and human PRMT5s, and the regions of the solved structures of rat PRMT1 and mouse CARM1 are aligned. Residues conserved in all four proteins are shown in white letters over purple background, and similar residues are indicated with red letters. Residues conserved in PRMT5 proteins and type-I arginine methylases are highlighted tan and yellow, respectively. Blue stars mark the CePRMT5 residues subjected to mutagenesis. At the top of the sequences, a schematic representation of the secondary structure elements of CePRMT5 is shown. Every ten residues are indicated with a “·” sign. (C) Enzymatic activity assay. Top box, coomassie-stained gel of enzymes and substrate (histone H4) used. GST-tagged rat PRMT1 and poly(His)-tagged C. elegans PRMT5 were expressed in E. coli, and flag-tagged human PRMT5 was purified from HEK293 cells. Approximately 5 μg of enzymes and histone H4 each were used in the assay. Top 2nd box, autoradiograph generated with the use of 0.25 mCi of SAM with tritiated methyl group. Top 3rd box, Western blot detection of asymmetrically dimethylated histone H4R3. Bottom box, Western blot detection of symmetrically dimethylated histone H4R3.