Shep RNA-Binding Capacity Is Required for Antagonism of gypsy Chromatin Insulator Activity

Abstract Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA–protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP- and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep associates with chromatin cotranscriptionally, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity.

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PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

Nucleic Acids Research ◽

10.1093/nar/gkaa211 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5511-5526

Author(s):

Tiago R Ferreira ◽

Adam A Dowle ◽

Ewan Parry ◽

Eliza V C Alves-Ferreira ◽

Karen Hogg ◽

...

Keyword(s):

Protein Modification ◽

Transcriptional Control ◽

Leishmania Major ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Capacity ◽

Arginine Methylation ◽

Mrna Metabolism

Abstract RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania 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 RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively 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 thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

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Functional Domain Structure of Human T-Cell Leukemia Virus Type 2 Rex

Journal of Virology ◽

10.1128/jvi.77.23.12829-12840.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12829-12840 ◽

Cited By ~ 15

Author(s):

Murli Narayan ◽

Ihab Younis ◽

Donna M. D'Agostino ◽

Patrick L. Green

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Binding Capacity ◽

Leukemia Virus ◽

Functional Domain ◽

Wild Type ◽

Human T Cell ◽

T Cell Leukemia Virus ◽

Carboxy Terminal

ABSTRACT The Rex protein of human T-cell leukemia virus (HTLV) acts posttranscriptionally to induce the cytoplasmic expression of the unspliced and incompletely spliced viral RNAs encoding the viral structural and enzymatic proteins and is therefore essential for efficient viral replication. Rex function requires nuclear import, RNA binding, multimerization, and nuclear export. In addition, it has been demonstrated that the phosphorylation status of HTLV-2 Rex (Rex-2) correlates with RNA binding and inhibition of splicing in vitro. Recent mutational analyses of Rex-2 revealed that the phosphorylation of serine residues 151 and 153 within a novel carboxy-terminal domain is critical for function in vivo. To further define the functional domain structure of Rex-2, we evaluated a panel of Rex-2 mutants for subcellular localization, RNA binding capacity, multimerization and trans-dominant properties, and the ability to shuttle between the nucleus and the cytoplasm. Rex-2 mutant S151A,S153A, which is defective in phosphorylation and function, showed diffuse cytoplasmic staining, whereas mutant S151D,S153D, previously shown to be functional and in a conformation corresponding to constitutive phosphorylation, displayed increased intense speckled staining in the nucleoli. In vivo RNA binding analyses indicated that mutant S151A,S153A failed to efficiently bind target RNA, while its phosphomimetic counterpart, S151D,S153D, bound twofold more RNA than wild-type Rex-2. Taken together, these findings provide direct evidence that the phosphorylation status of Rex-2 is linked to cellular trafficking and RNA binding capacity. Mutants with substitutions in either of the two putative multimerization domains or in the putative activation domain-nuclear export signal displayed a dominant negative phenotype as well as defects in multimerization and nucleocytoplasmic shuttling. Several carboxy-terminal mutants that displayed wild-type levels of phosphorylation and localized to the nucleolus were also partially impaired in shuttling. This is consistent with the hypothesis that the carboxy terminus of Rex-2 contains a novel domain that is required for efficient shuttling. This work thus provides a more detailed functional domain map of Rex-2 and further insight into its regulation of HTLV replication.

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The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner

Journal of Cell Science ◽

10.1242/jcs.151126 ◽

2014 ◽

Vol 127 (13) ◽

pp. 2956-2966 ◽

Cited By ~ 12

Author(s):

M. R. King ◽

L. H. Matzat ◽

R. K. Dale ◽

S. J. Lim ◽

E. P. Lei

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Tissue Specific ◽

Chromatin Insulator ◽

Insulator Function ◽

Specific Manner

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The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury

PLoS Pathogens ◽

10.1371/journal.ppat.1009494 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009494

Author(s):

Yingying Guo ◽

Meleana M. Hinchman ◽

Mercedes Lewandrowski ◽

Shaun T. Cross ◽

Danica M. Sutherland ◽

...

Keyword(s):

Viral Infection ◽

Viral Entry ◽

Post Infection ◽

Rna Binding ◽

Binding Capacity ◽

Binding Activity ◽

Dependent Manner ◽

Viral Protein Synthesis ◽

Granule Formation ◽

Dsrna Binding

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

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A global screening identifies chromatin-enriched RNA-binding proteins and the transcriptional regulatory activity of QKI5 during monocytic differentiation

Genome Biology ◽

10.1186/s13059-021-02508-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yue Ren ◽

Yue Huo ◽

Weiqian Li ◽

Manman He ◽

Siqi Liu ◽

...

Keyword(s):

Transcriptional Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Capacity ◽

Gene Promoter ◽

Transcriptional Activator ◽

Monocytic Differentiation ◽

Global Localization ◽

Transcriptional Regulatory

Abstract Background Cellular RNA-binding proteins (RBPs) have multiple roles in post-transcriptional control, and some are shown to bind DNA. However, the global localization and the general chromatin-binding ability of RBPs are not well-characterized and remain undefined in hematopoietic cells. Results We first provide a full view of RBPs’ distribution pattern in the nucleus and screen for chromatin-enriched RBPs (Che-RBPs) in different human cells. Subsequently, by generating ChIP-seq, CLIP-seq, and RNA-seq datasets and conducting combined analysis, the transcriptional regulatory potentials of certain hematopoietic Che-RBPs are predicted. From this analysis, quaking (QKI5) emerges as a potential transcriptional activator during monocytic differentiation. QKI5 is over-represented in gene promoter regions, independent of RNA or transcription factors. Furthermore, DNA-bound QKI5 activates the transcription of several critical monocytic differentiation-associated genes, including CXCL2, IL16, and PTPN6. Finally, we show that the differentiation-promoting activity of QKI5 is largely dependent on CXCL2, irrespective of its RNA-binding capacity. Conclusions Our study indicates that Che-RBPs are versatile factors that orchestrate gene expression in different cellular contexts, and identifies QKI5, a classic RBP regulating RNA processing, as a novel transcriptional activator during monocytic differentiation.

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Protein methyltransferase 7 deficiency in Leishmania major increases neutrophil associated pathology in murine model

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009230 ◽

2021 ◽

Vol 15 (3) ◽

pp. e0009230

Author(s):

Juliana Alcoforado Diniz ◽

Mariana M. Chaves ◽

Slavica Vaselek ◽

Rubens D. Miserani Magalhães ◽

Rafael Ricci-Azevedo ◽

...

Keyword(s):

Transcriptional Control ◽

Leishmania Major ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Capacity ◽

Sand Fly ◽

Parasite Development ◽

Post Translational Modification ◽

Phlebotomus Duboscqi ◽

The Impact

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.

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Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

10.1101/2021.03.24.436822 ◽

2021 ◽

Author(s):

Ting Cai ◽

Zhenbao Yu ◽

Zhen Wang ◽

Chen Liang ◽

Stephane Richard

Keyword(s):

Protein Function ◽

Rna Binding ◽

Binding Capacity ◽

Viral Proteins ◽

Arginine Methylation ◽

Host Protein ◽

Hek293 Cells ◽

Type I ◽

Dependent Manner ◽

N Protein

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5'-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

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An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2460-2466.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2460-2466 ◽

Cited By ~ 2

Author(s):

G Brewer

Keyword(s):

Cellular Response ◽

Mrna Decay ◽

Rna Binding ◽

Binding Capacity ◽

Proteinase K ◽

Rapid Turnover ◽

A Cell ◽

Degradation Activity ◽

Proteinase K Treatment

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.

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