Evolutionarily Divergent Type II Protein Arginine Methyltransferase in Trypanosoma brucei

Recombinant MBP-TbPRMT5 exhibits time- and concentration-dependent in vitro methyltransferase activity. (A) Coomassie-stained 10% SDS-PAGE gel showing the partial purification of recombinant TbPRMT5. TbPRMT5 was cloned and expressed as an N-terminal MBP fusion protein and partially purified by affinity chromatography over amylose resin. M, markers; PI, preinduced; I, induced; E, eluate from amylose column. (B) Recombinant MBP-TbPRMT5 exhibits concentration-dependent in vitro methyltransferase activity. In vitro methylation assays were performed in triplicate for 1 h at 36°C in the presence of 1 μM [methyl-³H]AdoMet, 5 μM His-RBP16, and increasing concentrations of MBP-TbPRMT5. The reactions were stopped with the addition of SDS sample buffer, resolved by 15% polyacrylamide SDS-PAGE, and analyzed by fluorography. Bands were quantified using Bio-Rad Quantity One imaging software. The background OD was subtracted and set to 0. The graph shows the mean ± standard deviation of triplicate experiments. A representative fluorograph and corresponding Coomassie-stained gel are shown above the graph. The positions of MBP-TbPRMT5 and His-RBP16 in the Coomassie-stained gel are indicated with arrows. (C) Time course of MBP-TbPRMT5 in vitro methyltransferase activity. In vitro methyltransferase assays were performed in triplicate at 36°C in the presence of 1 μM [methyl-³H]AdoMet, 3 μM His-RBP16, and 0.3 μM MBP-TbPRMT5. Aliquots were taken at the indicated time points, and the reactions were stopped with the addition of SDS sample buffer. The reactions were analyzed as described for panel B, except baseline OD values were normalized to the minimal value of triplicate experiments. Labels as in panel B.

Recombinant MBP-TbPRMT5 exhibits broad substrate specificity. MBP-TbPRMT5 exhibits in vitro methyltransferase activity toward a synthetic RG peptide, core histones H4 and H2A (arrows), myelin basic protein, and the trypanosome RNA binding protein, His-RBP16. For the core histone substrates, in vitro methylation assays were performed at 36°C for 1 h in the presence of 1 μM [methyl-³H]AdoMet, 10 μg calf thymus core histones, and 0.1 μM of either rat GST-PRMT1 or MBP-TbPRMT5. For the remaining substrates, in vitro methylation assays were performed at 36°C for 1 h in the presence of 1 μM [methyl-³H]AdoMet, 30 μM RG peptide or 1.2 μM of the indicated substrates, and 0.3 μM of either rat GST-PRMT1 or MBP-TbPRMT5. The reaction mixtures were resolved on 10 or 15% SDS-PAGE gels, and tritiated substrates were visualized by fluorography. Representative fluorographs and corresponding Coomassie-stained SDS-PAGE gels of triplicate experiments are shown. The positions of enzymes and substrates are indicated with asterisks and arrows, respectively. Note that a breakdown product of MBP-TbPRMT5 comigrates with myelin basic protein. The fluorographs correspond to the regions of the gels indicated above with arrows.

Recombinant MBP-TbPRMT5 catalyzes the formation of monomethylarginine and symmetric dimethylarginine in a substrate- and concentration-dependent manner. (A) Amino acid analysis of an RG peptide methylated in vitro by recombinant MBP-TbPRMT5. In vitro methylation assays were performed at 36°C for 1 or 24 h (far-right lane) in the presence of 1 μM [methyl-³H]AdoMet, 30 μM RG peptide substrate, and either 0.3 μM GST-PRMT1 or the indicated concentrations of MBP-TbPRMT5. Samples were acid hydrolyzed to free amino acids and resolved by TLC alongside 30 nmol of the indicated amino acid standards shown to the right, as described in Materials and Methods. (B) Amino acid analysis of methylated calf thymus core histones. Assays were performed for 16 h at 36°C in the presence of 1 μM [methyl-³H]AdoMet, 10 to 40 μg core histone substrate, and either 0.07 μM GST-PRMT1 or 0.13 μM MBP-TbPRMT5. The reactions were stopped and analyzed as described for panel A. (C) Amino acid analysis of methylated myelin basic protein. Assays were performed for 16 h at 36°C in the presence of 1 μM [methyl-³H]AdoMet, the indicated molar excess (X) of myelin basic protein substrate, and either 2.2 μM GST-PRMT1 or 0.5 μM MBP-TbPRMT5. The reactions were stopped and analyzed as described for panel A. (D) Amino acid analysis of methylated His-RBP16. Assays were performed for 16 h at 36°C in the presence of 1 μM [methyl-³H]AdoMet, the indicated molar excess (X) of His-RBP16 substrate, and either 0.05 μM GST-PRMT1 or 0.7 μM MBP-TbPRMT5. The reactions were stopped and analyzed as described for panel A.

Amino acid analysis detects monomethylarginine and symmetric dimethylarginine in core histones and RBP16. Purified MBP-TbPRMT5 was incubated with the methyl-accepting substrate core histones (A) or His-RBP16 (B) and analyzed by amino acid analysis as described in Materials and Methods. TCA-precipitated and acid-hydrolyzed samples were applied to a cation-exchange column with unlabeled MMA, aDMA, and sDMA standards. To detect ³H radioactivity (open diamonds), 200 μl of each fraction was mixed with 400 μl water and 5 ml fluor. Radioactivity was determined using a Beckman LS6500 counter as an average of three 3-min counting cycles. Unlabeled standards (closed squares) were detected with a ninhydrin assay using 100 μl of each fraction (66). As expected, the ³H radiolabel for sDMA and MMA migrated slightly ahead of the nonisotopically labeled standards due to a tritium isotope effect in the ion-exchange chromatography, where three tritium atoms replace three hydrogen atoms on each methyl group (35, 40).

TbPRMT5 is constitutively expressed in PF and BF life stages and localizes to the cytoplasm. (A) Northern blot analysis of TbPRMT5 expression in PF and BF life stages. Twenty micrograms of total RNA isolated from log-phase PF and BF parasites was resolved on a 1.5% formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized with a full-length TbPRMT5 antisense riboprobe. To normalize for loading, the membrane was stripped and hybridized with a 5′ end-labeled oligonucleotide probe complementary to tubulin mRNA. (B) TbPRMT5 localizes to the cytoplasm. To determine the subcellular localization of TbPRMT5, a PF cell line expressing TbPRMT5-TAP was subjected to subcellular fractionation, followed by immunoblot analysis. Fifteen micrograms of wild-type (WT) or TbPRMT5-TAP (WC) whole-cell extract was resolved by SDS-PAGE alongside equivalent microgram amounts of TbPRMT5-TAP cytoplasmic (Cyt) and nuclear (Nuc) extracts. The proteins were electroblotted to nitrocellulose, and the fractionation of TbPRMT5-TAP was analyzed using the TAP-specific PAP soluble-complex reagent. The efficiency of subcellular fractionation was assessed by Western blotting using antibodies against cytoplasmic (TbHsp70.4) and nuclear (RNAP CTD) proteins.

Analysis of in vivo TbPRMT5 protein complexes. (A) Glycerol gradient fractionation of TbPRMT5-TAP-containing complexes. Whole-cell extract prepared from PF trypanosomes ectopically expressing a TbPRMT5-TAP fusion protein was fractionated by centrifugation on a 5 to 20% glycerol gradient. The fractions were resolved by either denaturing polyacrylamide gel electrophoesis (SDS PAGE; top) or nondenaturing PAGE (native PAGE; bottom), and TbPRMT5-TAP-containing complexes were analyzed by immunoblotting using the TAP-specific PAP reagent. Twenty micrograms of whole-cell extract (WC) was analyzed by both methods for comparison. The sedimentation values and molecular masses of protein standards are indicated. (B) Tandem-affinity purification of native TbPRMT5 protein complexes. Wild type (W) or TbPRMT5-TAP (T) whole-cell extracts were fractionated by tandem-affinity purification over consecutive IgG-Sepharose and calmodulin resin columns. A silver-stained SDS-PAGE gel of TbPRMT5-CBP purification and complex composition is shown. TbPRMT5-CBP (arrow; 91.5 kDa) was eluted from IgG-Sepharose resin after TEV protease cleavage (TEV eluate), purified over calmodulin resin (Calm), and eluted under native conditions with EGTA (EGTA eluate). Major copurifying proteins present only in eluates of TAP-expressing cells (EGTA eluate; cf. lanes W and T) are indicated (asterisks). WC Supe, whole-cell supernatant; IgG FT, IgG-Sepharose resin flowthrough; TEV, TEV protease; Calm FT, calmodulin resin flowthrough.

The enzymatic properties of TbPRMT5-CBP purified from T. brucei are equivalent to those of recombinant enzyme. (A) Silver-stained SDS-PAGE gel showing the final eluates of TbPRMT5-CBP purified by tandem-affinity purification over IgG-Sepharose and calmodulin resins. Three separate purifications were performed. Prior to final elution, the eluates were washed with either 0.15, 0.5, or 1 M NaCl. The asterisks denote salt-resistant TbPRMT5-CBP-copurifying proteins. (B) TbPRMT5-CBP methylates His-RBP16, but not MBP-TBRGG1, in vitro. In vitro methylation assays were performed at 36°C for 1 h in the presence of 1 μM [methyl-³H]AdoMet, 1.4 μM substrate, and either 0.1 μM recombinant MBP-TbPRMT5 or 0.6 nM TbPRMT5-CBP washed with 0.15, 0.5, or 1 M NaCl prior to elution. The reaction mixtures were analyzed by SDS-PAGE (top) and fluorography (bottom). (C) Amino acid analysis of His-RBP16 methylated in vitro by TbPRMT5-CBP. In vitro methylation assays were performed at 36°C for 16 h in the presence of 0.9 μM [methyl-3H]AdoMet, 2 μM His-RBP16, and either 0.02 μM rat GST-PRMT1, 0.2 μM recombinant MBP-TbPRMT5, or 1.1 nM TbPRMT5-CBP washed prior to elution with 0.15, 0.5, or 1 M NaCl. The reactions were stopped by TCA precipitation, acid hydrolyzed to free amino acids, and analyzed by TLC, as described in Materials and Methods. All abbreviations are as in Fig. 4.