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Functional interplay between protein arginine methyltransferases in Trypanosoma brucei.

Lott K, Zhu L, Fisk JC, Tomasello DL, Read LK - Microbiologyopen (2014)

Bottom Line: Arginine methylation is a common posttranslational modification that has far-reaching cellular effects.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.Together, our studies indicate that TbPRMTs display a functional interplay at multiple levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University at Buffalo School of Medicine, Buffalo, New York, 14214.

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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.
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fig06: 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.

Mentions: Having shown that TbPRMT3 knockdown results in a similar overall TbPRMT profile as observed in TbPRMT1 knockdown cells (Fig.1), we predicted that the cellular methylation status in TbPRMT3 knockdown cells would be comparable to that seen in the TbPRMT1 knockdown cells. Western blot analysis showed that ADMA levels as detected by ASYM24 indeed exhibited a dramatic decrease similar to that observed in TbPRMT1 knockdown cells (compare Figs.2, 6). There appears to be no additional ADMA loss compared to that observed in TbPRMT1 depleted cells. We next monitored MMA levels in the TbPRMT3 RNAi cells. MMA in both the R*GG and MeR4 contexts dramatically increased upon PRMT3 depletion (Fig.6). The MMA pattern that emerged from TbPRMT3 depletion mimics that seen in the TbPRMT1 depletion, with no addition or loss of specific MMA signals. Given that the expression of TbPRMT1 and TbPRMT3 is mutually dependent and knockdown of either enzyme produces similar phenotypes, our data suggest that these enzymes form a functional hetero-oligomer in T. brucei, although further studies are needed to define this interaction.


Functional interplay between protein arginine methyltransferases in Trypanosoma brucei.

Lott K, Zhu L, Fisk JC, Tomasello DL, Read LK - Microbiologyopen (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234254&req=5

fig06: 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.
Mentions: Having shown that TbPRMT3 knockdown results in a similar overall TbPRMT profile as observed in TbPRMT1 knockdown cells (Fig.1), we predicted that the cellular methylation status in TbPRMT3 knockdown cells would be comparable to that seen in the TbPRMT1 knockdown cells. Western blot analysis showed that ADMA levels as detected by ASYM24 indeed exhibited a dramatic decrease similar to that observed in TbPRMT1 knockdown cells (compare Figs.2, 6). There appears to be no additional ADMA loss compared to that observed in TbPRMT1 depleted cells. We next monitored MMA levels in the TbPRMT3 RNAi cells. MMA in both the R*GG and MeR4 contexts dramatically increased upon PRMT3 depletion (Fig.6). The MMA pattern that emerged from TbPRMT3 depletion mimics that seen in the TbPRMT1 depletion, with no addition or loss of specific MMA signals. Given that the expression of TbPRMT1 and TbPRMT3 is mutually dependent and knockdown of either enzyme produces similar phenotypes, our data suggest that these enzymes form a functional hetero-oligomer in T. brucei, although further studies are needed to define this interaction.

Bottom Line: Arginine methylation is a common posttranslational modification that has far-reaching cellular effects.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.Together, our studies indicate that TbPRMTs display a functional interplay at multiple levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University at Buffalo School of Medicine, Buffalo, New York, 14214.

Show MeSH
Related in: MedlinePlus