Functional interplay between protein arginine methyltransferases in Trypanosoma brucei

Abstract

Arginine methylation is a common posttranslational modification that has far-reaching cellular effects. Trypanosoma brucei is an early-branching eukaryote with four characterized protein arginine methyltransferases (PRMTs), one additional putative PRMT, and over 800 arginine methylated proteins, suggesting that arginine methylation has widespread impacts in this organism. While much is known about the activities of individual T. brucei PRMTs (TbPRMTs), little is known regarding how TbPRMTs function together in vivo. In this study, we analyzed single and selected double TbPRMT knockdowns for the impact on expression of TbPRMTs and global methylation status. Repression of TbPRMT1 caused a decrease in asymmetric dimethylarginine and a marked increase in monomethylarginine that was catalyzed by TbPRMT7, suggesting that TbPRMT1 and TbPRMT7 can compete for the same substrate. We also observed an unexpected and strong interdependence between TbPRMT1 and TbPRMT3 protein levels. This finding, together with the observation of similar methyl landscape profiles in TbPRMT1 and TbPRMT3 repressed cells, strongly suggests that these two enzymes form a functional complex. We show that corepression of TbPRMT6/7 synergistically impacts growth of procyclic-form T. brucei. Our findings also implicate the actions of noncanonical, and as yet unidentified, PRMTs in T. brucei. Together, our studies indicate that TbPRMTs display a functional interplay at multiple levels.

Keywords: Arginine methylation; PRMTs; Trypanosomes; posttranslational modifications.

Figure 1

TbPRMT expression patterns upon TbPRMT1 repression. (A) Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT1 were harvested on day 3 or 4 postinduction, lysed in SDS-PAGE sample buffer, and resolved via 12.5% SDS-PAGE. Each lane contains 5 × 10⁶ cell equivalents. Western blot analysis was carried out using antibodies specific to each PRMT. Load controls shown under each blot. The protein p22 was used as a load control in all western blots except for the TbPRMT7 blot, in which the mitochondrial protein, TbRGG2 was used. Quantification of TbPRMTs is represented as normalized percentages compared to TbPRMT1-expressing cells. (B) Quantitative RT-PCR analysis of TbPRMT1, TbPRMT3, and TbPRMT6 RNA levels in TbPRMT1-repressed cells relative to those in TbPRMT1-expressing cells. RNA levels were normalized against 18S rRNA and represent the average and SD of 3–6 determinations.

Figure 2

TbPRMT1 repression leads to a decrease in ADMA and an increase in MMA. Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT1 were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 1 × 10⁷ cell equivalents. Western blot analysis was carried out using three different antimethylarginine antibodies: ASYM24 detects ADMA; MMA(R*GG) and MMA(MeR⁴) detect MMA. p22 (bottom) is shown as a load control.

Figure 3

Expression patterns of TbPRMTs upon simultaneous TbPRMT1/7 knockdown. (A) Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT1/7 were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 5 × 10⁶ cell equivalents. Western blot analysis was carried out using antibodies specific to each PRMT. Load controls shown under each blot. The protein p22 was used as a load control in all western blots except for the TbPRMT7 blot, in which the mitochondrial protein, TbRGG2, was used. Quantification of TbPRMTs is represented as normalized percentages compared to TbPRMT1/7-expressing cells. (B) Quantitative RT-PCR analysis of TbPRMT1, TbPRMT7, TbPRMT3, and TbPRMT6 RNA levels in TbPRMT1/7-repressed cells relative to those in TbPRMT1/7-expressing cells. RNA levels were normalized against 18S rRNA and represent the average and SD of 3–6 determinations.

Figure 4

Simultaneous TbPRMT1/7 repression reveals substrate scavenging by TbPRMT7. Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT1, or both TbPRMT1 and TbPRMT7 (TbPRMT1/7), were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 1 × 10⁷ cell equivalents. Western blot analysis was carried out using three different antimethylarginine antibodies: ASYM24 detects ADMA, and MMA(R*GG) and MMA(MeR⁴) detect MMA. The p22 load control is shown under each blot.

Figure 5

TbPRMT3 repression leads to a decrease in TbPRMT1 protein levels. (A) Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT3 were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 5 × 10⁶ cell equivalents. Western blot analysis was carried out using antibodies specific to each TbPRMT. p22 serves as a load control and is shown below each respective TbPRMT western blot. Quantification of TbPRMTs is represented as normalized percentages compared to TbPRMT3-expressing cells. (B) Quantitative RT-PCR analysis of TbPRMT1, TbPRMT3, and TbPRMT6 RNAs in TbPRMT3-repressed cells relative to those in TbPRMT3-expressing cells. RNA levels were normalized against 18S rRNA and represent the average and SD of 3–6 determinations.

Figure 6

Cells repressed for TbPRMT3 display the same methyl landscape as cells repressed for TbPRMT1. Trypanosoma brucei procyclic cells either expressing (E) or repressed (R) for TbPRMT3 were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 1 × 10⁷ cell equivalents. Western blot analysis was carried out using three different antimethylarginine antibodies: ASYM24 detects ADMA, and MMA(R*GG) and MMA(MeR⁴) detect MMA. The p22 load control is shown under each blot.

Figure 7

TbPRMT7 does not appear to scavenge TbPRMT6 substrates. (A) Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT6 or both TbPRMT6 and TbPRMT7 (TbPRMT6/7) were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 5 × 10⁶ cell equivalents. Western blot analysis was carried out using antibodies specific to each TbPRMT. p22 serves as the load control and is shown below each respective western blot. Quantification of bands is represented as normalized percentages compared to PRMT-expressing cells. (B) Western blot analysis was carried out on cell lines from above using three different antimethyl antibodies: ASYM24 detects ADMA, and MMA(R*GG) and MMA(MeR⁴) detect MMA. The p22 is a load control shown under each blot.

Figure 8

Simultaneous depletion of TbPRMT6 and TbPRMT7 impairs parasite growth. Trypanosoma brucei procyclic-form cells either expressing (solid black line) or repressed for (dotted grey line) TbPRMT6 and TbPRMT7 were assessed for parasite growth over the course of 14 days. Cells from triplicate cultures were counted every 2 days and diluted back to a starting concentration of 8 × 10⁵ cells/mL. Error bars represent standard deviation from triplicate experiments.

Figure 9

TbPRMT7 repression does not detectably alter MMA levels. (A) Trypanosoma brucei procyclic-form cells either expressing (E) or repressed (R) for TbPRMT7 were lysed in SDS-PAGE sample buffer and resolved via 12.5% SDS-PAGE. Each lane contains 5 × 10⁶ cell equivalents. Western blot analysis was carried out using antibodies specific to each PRMT. The p22 load control is shown below each blot. Quantification of bands is represented as normalized percentages compared to PRMT-expressing cells. (B) Western blot analysis was carried out on cell lines shown in 9A using three different antimethylarginine antibodies: ASYM24 detects ADMA, and MMA(R*GG) and MMA(MeR⁴) detect MMA. (C) Western blot analysis of steady-state MMA levels over a 22-day time course following TbPRMT7 repression. The last lane in each blot (22-) indicates cells grown under the same conditions for 22 days in the absence of tetracycline addition. The p22 load control is shown under each blot.