Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

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.

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Genomic and cDNA selection-amplification identifies transcriptome-wide binding sites for the Drosophila protein sex-lethal

PLoS ONE ◽

10.1371/journal.pone.0250592 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0250592

Author(s):

Hiren Banerjee ◽

Ravinder Singh

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Developmental Stages ◽

Rna Binding Proteins ◽

Systematic Analysis ◽

Downstream Targets ◽

Drosophila Protein ◽

Sex Lethal ◽

Female Specific

Background Downstream targets for a large number of RNA-binding proteins remain to be identified. The Drosophila master sex-switch protein Sex-lethal (SXL) is an RNA-binding protein that controls splicing, polyadenylation, or translation of certain mRNAs to mediate female-specific sexual differentiation. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens. Methodology/Principal findings Here, we have used an alternative molecular approach of GEnomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) using both the genomic DNA and cDNA pools from several Drosophila developmental stages to identify new potential targets of SXL. Our systematic analysis provides a comprehensive view of the Drosophila transcriptome for potential SXL-binding sites. Conclusion/Significance We have successfully identified new SXL-binding sites in the Drosophila transcriptome. We discuss the significance of our analysis and that the newly identified binding sites and sequences could serve as a useful resource for the research community. This approach should also be applicable to other RNA-binding proteins for which downstream targets are unknown.

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

Biological Chemistry ◽

10.1515/hsz-2013-0121 ◽

2013 ◽

Vol 394 (8) ◽

pp. 1029-1043 ◽

Cited By ~ 3

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.

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In vivo and in vitro arginine methylation of RNA-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.15.5.2800 ◽

1995 ◽

Vol 15 (5) ◽

pp. 2800-2808 ◽

Cited By ~ 214

Author(s):

Q Liu ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Posttranslational Modifications ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Arginine Methylation ◽

Hnrnp A1 ◽

Arginine Residues

Heterogenous nuclear ribonucleoproteins (hnRNPs) bind pre-mRNAs and facilitate their processing into mRNAs. Many of the hnRNPs undergo extensive posttranslational modifications including methylation on arginine residues. hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. The role of this modification is not known. Here we identify the hnRNPs that are methylated in HeLa cells and demonstrate that most of the pre-mRNA-binding proteins receive this modification. Using recombinant human hnRNP A1 as a substrate, we have partially purified and characterized a protein-arginine N-methyltransferase specific for hnRNPs from HeLa cells. This methyltransferase can methylate the same subset of hnRNPs in vitro as are methylated in vivo. Furthermore, it can also methylate other RNA-binding proteins that contain the RGG motif RNA-binding domain. This activity is evolutionarily conserved from lower eukaryotes to mammals, suggesting that methylation has a significant role in the function of RNA-binding proteins.

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

10.1101/2021.09.22.461376 ◽

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.

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Post-translational epigenetics: PRMT7 regulates RNA-binding capacity and protein stability to controlLeishmaniaparasite virulence

10.1101/736736 ◽

2019 ◽

Author(s):

Tiago R. Ferreira ◽

Adam A. Dowle ◽

Ewan Parry ◽

Eliza V. C. Alves-Ferreira ◽

Foteini Kolokousi ◽

...

Keyword(s):

Protein Modification ◽

Transcriptional Control ◽

Leishmania Major ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Capacity ◽

Arginine Methylation ◽

Protein Arginine Methyltransferases

ABSTRACTRNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, makingLeishmaniaan exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine MethylTransferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. InLeishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substratesin vitro. The absence of PRMT7 levelsin vivoselectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and stability ofδ-amastinsurface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation upon RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence inLeishmania. This work introducesLeishmaniaPRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

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The Role of Protein Arginine Methylation in mRNP Dynamics

Molecular Biology International ◽

10.4061/2011/163827 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 28

Author(s):

Michael C. Yu

Keyword(s):

Messenger Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Arginine Methylation ◽

Messenger Ribonucleoprotein ◽

Heterogeneous Nuclear Ribonucleoproteins ◽

Rna Biogenesis ◽

Mrnp Biogenesis

In eukaryotes, messenger RNA biogenesis depends on the ordered and precise assembly of a nuclear messenger ribonucleoprotein particle (mRNP) during transcription. This process requires a well-orchestrated and dynamic sequence of molecular recognition events by specific RNA-binding proteins. Arginine methylation is a posttranslational modification found in a plethora of RNA-binding proteins responsible for mRNP biogenesis. These RNA-binding proteins include both heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins. In this paper, I discuss the mechanisms of action by which arginine methylation modulates various facets of mRNP biogenesis, and how the collective consequences of this modification impart the specificity required to generate a mature, translational- and export-competent mRNP.

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Systematic Analysis of the Impact of R-Methylation on RBPs-RNA Interactions: A Proteomic Approach

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.688973 ◽

2021 ◽

Vol 8 ◽

Author(s):

Marianna Maniaci ◽

Francesca Ludovica Boffo ◽

Enrico Massignani ◽

Tiziana Bonaldi

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Molecular Complexes ◽

Global Changes ◽

Type I ◽

Stable Isotope Labelling ◽

Systematic Analysis ◽

Protein Arginine Methyltransferases ◽

Proteomic Approach ◽

The Impact

RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-molecular complexes known as ribonucleoproteins (RNPs). Various post-translational modifications (PTMs) have been described to regulate RBP structure, subcellular localization, and interactions with other proteins or RNAs. Recent proteome-wide experiments showed that RBPs are the most representative group within the class of arginine (R)-methylated proteins. Moreover, emerging evidence suggests that this modification plays a role in the regulation of RBP-RNA interactions. Nevertheless, a systematic analysis of how changes in protein-R-methylation can affect globally RBPs-RNA interactions is still missing. We describe here a quantitative proteomics approach to profile global changes of RBP-RNA interactions upon the modulation of type I and II protein arginine methyltransferases (PRMTs). By coupling the recently described Orthogonal Organic Phase Separation (OOPS) strategy with the Stable Isotope Labelling with Amino acids in Cell culture (SILAC) and pharmacological modulation of PRMTs, we profiled RNA-protein interaction dynamics in dependence of protein-R-methylation. Data are available via ProteomeXchange with identifier PXD024601.

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Protein Arginine Methylation in Candida albicans: Role in Nuclear Transport

Eukaryotic Cell ◽

10.1128/ec.00074-07 ◽

2007 ◽

Vol 6 (7) ◽

pp. 1119-1129 ◽

Cited By ~ 26

Author(s):

Anne E. McBride ◽

Cecilia Zurita-Lopez ◽

Anthony Regis ◽

Emily Blum ◽

Ana Conboy ◽

...

Keyword(s):

Candida Albicans ◽

Protein Transport ◽

Rna Binding ◽

Fluorescent Protein ◽

Rna Binding Proteins ◽

Nuclear Transport ◽

Arginine Methylation ◽

Type I ◽

Sequencing Project ◽

Protein Arginine Methylation

ABSTRACT Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and ω-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Δ strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Δ/rmt2Δ strain, as well as biochemical evidence for additional cryptic PRMTs.

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A systematic survey of PRMT interactomes reveals key roles of arginine methylation in the global control of RNA splicing and translation

10.1101/746529 ◽

2019 ◽

Cited By ~ 2

Author(s):

Huan-Huan Wei ◽

Xiao-Juan Fan ◽

Yue Hu ◽

Xiao-Xu Tian ◽

Meng Guo ◽

...

Keyword(s):

Rna Splicing ◽

Ribosomal Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Arginine Methylation ◽

Mrna Splicing ◽

Post Translational Modification ◽

Protein Arginine Methyltransferases ◽

Full Picture ◽

Catalytic Network

AbstractThousands of proteins undergo arginine methylation, a widespread post-translational modification catalyzed by various protein arginine methyltransferases (PRMTs). However, a full picture of the catalytic network for each PRMT is lacking and the global understanding of their biological roles remains limited. Here we systematically identified interacting proteins for all human PRMTs and demonstrated that they are functionally important for mRNA splicing and translation. We showed that the interactomes of human PRMTs are significantly overlapped with the known methylarginine containing proteins, and different PRMTs are functionally complementary with a high degree of overlap in their substrates and high similarities between their putative methylation motifs. Importantly, arginine methylation is significantly enriched in RNA binding proteins involved in regulating RNA splicing and translation, and inhibition of PRMTs leads to global alteration of alternative splicing and suppression of translation. In particular, ribosomal proteins are pervasively modified with methylarginine, and mutations on their methylation sites suppress ribosome assembly, translation, and eventually cell growth. Collectively, our study provides a novel global view of different PRMT networks and uncovers critical functions of arginine methylation in the regulation of mRNA splicing and translation.

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Altered RNA splicing initiates the viral mimicry response from inverted SINEs following type I PRMT inhibition in Triple-Negative Breast Cancer

10.21203/rs.3.rs-649284/v1 ◽

2021 ◽

Author(s):

Cheryl Arrowsmith ◽

Qin Wu ◽

David Nie ◽

Wail alawi ◽

Jennifer Cruickshank ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Rna Binding ◽

Rna Binding Proteins ◽

Breast Cancer Subtype ◽

Arginine Methylation ◽

Interferon Response ◽

Type I

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here, we undertook a screen of epigenetic chemical probes to systematically uncover the epigenetic regulators critical for TNBC growth. We identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), as having anti-tumor growth activity in TNBC in vitro and in vivo. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses pre- and post-MS023 treatment is a functional biomarker and determinant of response; and these observations extend to a panel of patient-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA (dsRNA). The observed dsRNA accumulation is derived, at least in part, from inverted-repeat Alus (IR-Alus), many of which are expressed from retained introns induced by MS023, which inhibits arginine methylation of RNA-binding proteins and alters mRNA splicing machinery. Together, our results represent a shift in understanding the anti-tumor mechanism of type I PRMT inhibitors and provide a novel rationale and biomarker approach for the clinical development of type I PRMT inhibitors.

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