Comparative Monomethylarginine Proteomics Suggests that Protein Arginine Methyltransferase 1 (PRMT1) is a Significant Contributor to Arginine Monomethylation in Toxoplasma gondii

Abstract

Arginine methylation is a common posttranslational modification found on nuclear and cytoplasmic proteins that has roles in transcriptional regulation, RNA metabolism and DNA repair. The protozoan parasite has a complex life cycle requiring transcriptional plasticity and has unique transcriptional regulatory pathways. Arginine methylation may play an important part in transcriptional regulation and splicing biology in this organism. The genome contains five putative protein arginine methyltransferases (PRMTs), of which PRMT1 is important for cell division and growth. In order to better understand the function(s) of the posttranslational modification monomethyl arginine (MMA) in , we performed a proteomic analysis of MMA proteins using affinity purification employing anti-MMA specific antibodies followed by mass spectrometry. The arginine monomethylome of contains a large number of RNA binding proteins and multiple ApiAP2 transcription factors, suggesting a role for arginine methylation in RNA biology and transcriptional regulation. Surprisingly, 90% of proteins that are arginine monomethylated were detected as being phosphorylated in a previous phosphoproteomics study which raises the possibility of interplay between MMA and phosphorylation in this organism. Supporting this, a number of kinases are also arginine methylated. Because PRMT1 is thought to be a major PRMT in , an organism which lacks a MMA-specific PRMT, we applied comparative proteomics to understand how PRMT1 might contribute to the MMA proteome in We identified numerous putative PRMT1 substrates, which include RNA binding proteins, transcriptional regulators ( AP2 transcription factors), and kinases. Together, these data highlight the importance of MMA and PRMT1 in arginine methylation in , as a potential regulator of a large number of processes including RNA biology and transcription.

Post‐translational modifications as key regulators of apicomplexan biology: insights from proteome‐wide studies

“…Recently, the significance of arginine methylation as a dynamic modification has emerged. Arginine monomethylated (MMA) proteins are involved in a wide range of functions and enriched in DNA and RNA binding proteins, many of which are highly modified by up to 7 MMA sites (Yakubu et al ., ). The MMA proteome covers almost 5% of the T. gondii proteome (Yakubu et al ., ).…”

“…Arginine monomethylated (MMA) proteins are involved in a wide range of functions and enriched in DNA and RNA binding proteins, many of which are highly modified by up to 7 MMA sites (Yakubu et al ., ). The MMA proteome covers almost 5% of the T. gondii proteome (Yakubu et al ., ). Ubiquitination was detected as being similarly abundant, on up to 5% of the T. gondii or P. falciparum predicted proteomes (Ponts et al ., ; Silmon de Monerri et al ., ).…”

“…The motor complex is the core component responsible for parasite motility and host cell invasion (Meissner et al ., ) and consists of myosin heavy chain protein TgMyoA (Herm‐Gotz et al ., ; Nebl et al ., ), two myosin light chains: TgMLC1, TgELC1 and gliding associated proteins TgGAP45 and TgGAP50 (Frenal et al ., ). The motor complex is highly modified by palmitoylation, ubiquitin, phosphorylation and methylated on, for example, myosin heavy chain and myosin E, F and J (Yakubu et al ., ). Ubiquitination has been found on some of the glidosome proteins (TgGAP40, TgGAP45, TgGAP50, TgMLC1 and several myosins), and has been speculated to be important for rearrangement of the cytoskeleton as it is abundant on cytoskeletal and structural proteins in T. gondii (Silmon de Monerri et al ., ).…”

“…In eukaryotes, ubiquitination of tubulin regulates tubulin polymerization and inhibits microtubule formation (Bheda et al ., ); it may have similar regulatory effects on parasite tubulin. Several methylated IMC proteins were also found in an examination of the T. gondii monomethylome including IMC1, IMC4 and SPM2 (Yakubu et al ., ). Similarly, three phosphorylated serines on TgISP2, two phosphorylated serines on TgISP3 and phosphorylation of PfISP3 were identified in the T. gondii and P. falciparum phosphoproteomes (Treeck et al ., ).…”

“…In T. gondii five ApiAP2 (AP2IX‐5, AP2IX‐7, AP2X‐5, AP2XII‐4 and AP2XII‐8), have acetylation sites that lie outside of the AP2 DNA binding domain and one acetyl‐lysine at K570 is found within the first AP2 DNA binding domain on AP2X‐7 (TGME49_014840) (Jeffers and Sullivan, ). Similar patterns are found with regard to arginine monomethylation, where this PTM has been found outside of the AP2 DNA‐binding domain in ten ApiAP2 proteins (Yakubu et al ., ). Ubiquitination sites have been found in five ApiAP2 in T. gondii (Silmon de Monerri et al ., ), with only AP2X‐7 (TGME49_214840) containing ubiquitin (at K1368) within its third ApiAP2 domain.…”