scholarly journals Functional study of Leishmania braziliensis protein arginine methyltransferases (PRMTs) reveals that PRMT1 and PRMT5 are required for macrophage infection

2021 ◽  
Author(s):  
Lucas Lorenzon ◽  
Jose Carlos Quilles ◽  
Gustavo Daniel Campagnaro ◽  
Leticia Almeida ◽  
Flavio Protasio Veras ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Rna Binding ◽  
Arginine Methylation ◽  
Functional Study ◽  
Leishmania Braziliensis ◽  
Macrophage Infection ◽  
Protein Arginine Methyltransferases ◽  
Life Cycle Stages ◽  
Arginine Residues

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are major targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other macromolecular interactions of RBPs by transferring methyl groups to protein arginine residues. Herein, we present the results of a study that functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that arginine methylation profiles vary among Leishmania species and that target protein methylation changes across different L. braziliensis life cycle stages, with higher PRMT expression in the promastigote stages than in the axenic amastigote stage. Knockout of some of the L. braziliensis PRMTs led to significant changes in global arginine methylation patterns without affecting promastigote axenic growth. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine (MMA) levels, which is mainly catalyzed by PRMT7. Putative targets and/or PRMT-interacting proteins were identified by coimmunoprecipitation using HA-tagged PRMTs, revealing a network of target RBPs and suggesting functional interactions between them and a relevant participation in epigenetic control of gene expression. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and that in the absence of PRMT1 and PRMT5, axenic amastigote proliferation is impaired. The results indicate that arginine methylation is modulated across life cycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

scholarly journals Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

2004 ◽  
Vol 379 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Marie-Chloé BOULANGER ◽  
Tina Branscombe MIRANDA ◽  
Steven CLARKE ◽  
Marco di FRUSCIO ◽  
Beat SUTER ◽  
...  
Keyword(s):  
Rna Binding ◽  
Developmental Stages ◽  
Rna Binding Proteins ◽  
Genetic System ◽  
Arginine Methylation ◽  
Type I ◽  
Protein Arginine Methyltransferases ◽  
Arginine Residues ◽  
Drosophila Protein

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 (Drosophilaarginine methyltransferases 1–9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

scholarly journals Prmt7 is dispensable in tissue culture models for adipogenic differentiation

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 279 ◽  
Author(s):  
Yu-Jie Hu ◽  
Saïd Sif ◽  
Anthony N. Imbalzano
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Adipogenic Differentiation ◽  
Arginine Methylation ◽  
Type Ii ◽  
Protein Arginine Methyltransferases ◽  
Over Expression ◽  
Core Histones ◽  
Culture Models ◽  
Arginine Residues

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

scholarly journals Arginine Methyltransferases as Regulators of RNA-Binding Protein Activities in Pathogenic Kinetoplastids

2021 ◽  
Vol 8 ◽  
Author(s):  
Gustavo D. Campagnaro ◽  
Edward Nay ◽  
Michael J. Plevin ◽  
Angela K. Cruz ◽  
Pegine B. Walrad
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Structural Features ◽  
Arginine Methylation ◽  
Diverse Range ◽  
Host Interaction ◽  
Intronless Genes

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a “regulatory code”. Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality.

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

2013 ◽  
Vol 394 (8) ◽  
pp. 1029-1043 ◽  
Author(s):  
Elmar Wahle ◽  
Bodo Moritz
Keyword(s):  
Active Sites ◽  
Binding Protein ◽  
Arginine Methylation ◽  
Fine Tuning ◽  
General Mechanism ◽  
Type I ◽  
Protein Arginine Methyltransferases ◽  
Low Protein ◽  
Backbone Conformation ◽  
Arginine Residues

Abstract Asymmetric dimethylation of arginine side chains in proteins is a frequent posttranslational modification, catalyzed by type I protein arginine methyltransferases (PRMTs). This article summarizes what is known about this modification in the nuclear poly(A)-binding protein (PABPN1). PABPN1 contains 13 dimethylated arginine residues in its C-terminal domain. Three enzymes, PRMT1, 3, and 6, can methylate PABPN1. Although 26 methyl groups are transferred to one PABPN1 molecule, the PRMTs do so in a distributive reaction, i.e., only a single methyl group is transferred per binding event. As PRMTs form dimers, with the active sites accessible from a small central cavity, backbone conformation around the methyl-accepting arginine is an important determinant of substrate specificity. Neither the association of PABPN1 with poly(A) nor its role in poly(A) tail synthesis is affected by arginine methylation. At least at low protein concentration, methylation does not affect the protein’s tendency to oligomerize. The dimethylarginine residues of PABPN1 are located in the binding site for its nuclear import receptor, transportin. Arginine methylation weakens this interaction about 10-fold. Very recent evidence suggests that arginine methylation as a way of fine-tuning the interactions between transportin and its cargo may be a general mechanism.

scholarly journals Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gilbert O. Silveira ◽  
Murilo S. Amaral ◽  
Helena S. Coelho ◽  
Lucas F. Maciel ◽  
Adriana S. A. Pereira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Schistosoma Mansoni ◽  
Reference Genes ◽  
Large Scale ◽  
Developmental Stages ◽  
Adult Males ◽  
Histone H4 ◽  
Expression Levels ◽  
Life Cycle Stages

AbstractReverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.

scholarly journals Protein Arginine Methylation in Parasitic Protozoa

2011 ◽  
Vol 10 (8) ◽  
pp. 1013-1022 ◽  
Author(s):  
John C. Fisk ◽  
Laurie K. Read
Keyword(s):  
Rna Processing ◽  
Antigenic Variation ◽  
Arginine Methylation ◽  
Parasitic Protozoa ◽  
Content Type ◽  
Protein Arginine Methyltransferases ◽  
Functional Studies ◽  
Dna Replication And Repair ◽  
Arginine Residues ◽  
Multiple Species

ABSTRACT Protozoa constitute the earliest branch of the eukaryotic lineage, and several groups of protozoans are serious parasites of humans and other animals. Better understanding of biochemical pathways that are either in common with or divergent from those of higher eukaryotes is integral in the defense against these parasites. In yeast and humans, the posttranslational methylation of arginine residues in proteins affects myriad cellular processes, including transcription, RNA processing, DNA replication and repair, and signal transduction. The protein arginine methyltransferases (PRMTs) that catalyze these reactions, which are unique to the eukaryotic kingdom of organisms, first become evident in protozoa. In this review, we focus on the current understanding of arginine methylation in multiple species of parasitic protozoa, including Trichomonas , Entamoeba , Toxoplasma , Plasmodium , and Trypanosoma spp., and discuss how arginine methylation may play important and unique roles in each type of parasite. We mine available genomic and transcriptomic data to inventory the families of PRMTs in different parasites and the changes in their abundance during the life cycle. We further review the limited functional studies on the roles of arginine methylation in parasites, including epigenetic regulation in Apicomplexa and RNA processing in trypanosomes. Interestingly, each of the parasites considered herein has significantly differing sets of PRMTs, and we speculate on the importance of this diversity in aspects of parasite biology, such as differentiation and antigenic variation.

scholarly journals A Trypanosoma cruzi zinc finger protein that is implicated in the control of epimastigote-specific gene expression and metacyclogenesis

Parasitology ◽  
2020 ◽  
pp. 1-15
Author(s):  
Thais S. Tavares ◽  
Fernanda L. B. Mügge ◽  
Viviane Grazielle-Silva ◽  
Bruna M. Valente ◽  
Wanessa M. Goes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Trypanosoma Cruzi ◽  
Zinc Finger ◽  
Rna Binding ◽  
Environmental Changes ◽  
Negative Regulator ◽  
Specific Gene ◽  
Genes Encoding ◽  
Transcriptome Analyses

Abstract Trypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programmed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and have their expression controlled by post-transcriptional mechanisms. Transcriptome analyses comparing three stages of the T. cruzi life cycle revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBPs), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP, named TcZH3H12, which contains a zinc finger domain and is up-regulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout (KO) epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Transcriptome analyses comparing wild type and TcZC3H12 KOs revealed a TcZC3H12-dependent expression of epimastigote-specific genes such as genes encoding amino acid transporters and proteins associated with differentiation (PADs). RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

scholarly journals Analysis of Pir Gene Expression Across the Plasmodium Life Cycle

2021 ◽  
Author(s):  
Timothy S. Little ◽  
Deirdre A. Cunningham ◽  
Audrey Vandomme ◽  
Carlos Talavera Lopez ◽  
Sarah I. Amis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Antigenic Variation ◽  
Expression Patterns ◽  
Plasmodium Species ◽  
Data Sets ◽  
Life Cycle Stages ◽  
Determine Function ◽  
Gene Expression Studies ◽  
Functional Investigation

Abstract Background Plasmodium interspersed repeat ( pir ) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. Methods Here we examined pir gene expression across the life cycle of P. berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from P. chabaudi . Results Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. Conclusions By identifying distinct expression patterns for different pir genes and subfamilies we hope to provide a basis for the design of future experiments to uncover their function.

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