The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Masoud Vedadi - One of the best experts on this subject based on the ideXlab platform.
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a suite of biochemical assays for screening RNA Methyltransferase bcdin3d
Journal of Biomolecular Screening, 2017Co-Authors: Levi L Blazer, Fengling Li, Steven Kennedy, Yujun George Zheng, C H Arrowsmith, Masoud VedadiAbstract:BCDIN3D is an RNA-Methyltransferase that O-methylates the 5′ phosphate of RNA and regulates microRNA maturation. To discover small-molecule inhibitors of BCDIN3D, a suite of biochemical assays was developed. A radiometric Methyltransferase assay and fluorescence polarization–based S-adenosylmethionine and RNA displacement assays are described. In addition, differential scanning fluorimetry and surface plasmon resonance were used to characterize binding. These assays provide a comprehensive package for the development of small-molecule modulators of BCDIN3D activity.
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a radioactivity based assay for screening human m6a RNA Methyltransferase mettl3 mettl14 complex and demethylase alkbh5
Journal of Biomolecular Screening, 2016Co-Authors: Fengling Li, Steven Kennedy, C H Arrowsmith, Masoud Vedadi, Taraneh Hajian, Elisa Gibson, A Seitova, Chao XuAbstract:N6-methyladenosine (m6A) is the most common reversible inteRNAl modification in mammalian RNA. Changes in m6A levels have been implicated in a variety of cellular processes, including nuclear RNA export, control of protein translation, and protein splicing, and they have been linked to obesity, cancer, and other human diseases. METTL3 and METTL14 are N6-adenosine Methyltransferases that work more efficiently in a stable METTL3-METTL14 heterodimer complex (METTL3-14). ALKBH5 is an m6A-RNA demethylase that belongs to the AlkB family of dioxygenases. We report the development of radioactivity-based assays for kinetic characterization of m6A-RNA modifications by METTL3-14 complex and ALKBH5 and provide optimal assay conditions suitable for screening for ligands in a 384-well format with Z′ factors of 0.78 and 0.77, respectively.
Michaela Frye - One of the best experts on this subject based on the ideXlab platform.
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the mouse cytosine 5 RNA Methyltransferase nsun2 is a component of the chromatoid body and required for testis differentiation
Molecular and Cellular Biology, 2013Co-Authors: Shobbir Hussain, Sandra Blanco, Francesca Tuorto, Suraj Menon, Joana V Flores, Stephen Watt, Nobuaki Kudo, Frank Lyko, Michaela FryeAbstract:Posttranscriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and are often organized by the chromatoid body. Here, we identify the RNA Methyltransferase NSun2 as a novel component of the chromatoid body and, further, show that NSun2 is essential for germ cell differentiation in the mouse testis. In NSun2-depleted testes, genes encoding Ddx4, Miwi, and Tudor domain-containing (Tdr) proteins are repressed, indicating that RNA-processing and posttranscriptional pathways are impaired. Loss of NSun2 specifically blocked meiotic progression of germ cells into the pachytene stage, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 and Dnmt2, the only other cytosine-5 RNA Methyltransferase characterized to date, indicating that Dnmt2 was not functionally redundant with NSun2 in spermatogonial stem cells or Sertoli cells. Specific NSun2- and Dnmt2-methylated tRNAs decreased in abundance when both Methyltransferases were deleted, suggesting that RNA methylation pathways play an essential role in male germ cell differentiation.
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the RNA Methyltransferase misu nsun2 poises epidermal stem cells to differentiate
PLOS Genetics, 2011Co-Authors: Sandra Blanco, Agata Kurowski, Jennifer Nichols, Fiona M Watt, Salvador Aznar Benitah, Michaela FryeAbstract:Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA Methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this Methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate.
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the RNA Methyltransferase misu nsun2 mediates myc induced proliferation and is upregulated in tumors
Current Biology, 2006Co-Authors: Michaela Frye, Fiona M WattAbstract:Summary Background Myc is a well-known proto-oncogene, but its functions in normal tissue remain enigmatic. In adult epidermis, Myc stimulates exit from the stem cell compartment, decreasing cell adhesion and, by an unknown mechanism, triggering proliferation of transit-amplifying cells. Results We describe a novel direct target gene of Myc, Misu, that is expressed at low levels in normal epidermis but is upregulated on Myc activation. Misu encodes a previously uncharacterized RNA Methyltransferase with high sequence homology to NSun2 and defines a new family of mammalian SUN-domain-containing proteins. The nucleolar localization of Misu is dependent on RNA polymerase III transcripts, and knockdown of Misu decreases nucleolar size. In G2 phase of the cell cycle, Misu is found in cytoplasmic vesicles, and it decorates the spindle in mitosis. Misu expression is highest in S phase, and RNAi constructs block Myc-induced keratinocyte proliferation and cell-cycle progression. Misu is expressed at low levels in normal tissues, but is highly induced in a range of tumors. Growth of human squamous-cell-carcinoma xenografts is decreased by Misu RNAi. Conclusions Misu is a novel downstream Myc target that methylates RNA polymerase III transcripts. Misu mediates Myc-induced cell proliferation and growth and is a potential target for cancer therapies.
Shobbir Hussain - One of the best experts on this subject based on the ideXlab platform.
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the mouse cytosine 5 RNA Methyltransferase nsun2 is a component of the chromatoid body and required for testis differentiation
Molecular and Cellular Biology, 2013Co-Authors: Shobbir Hussain, Sandra Blanco, Francesca Tuorto, Suraj Menon, Joana V Flores, Stephen Watt, Nobuaki Kudo, Frank Lyko, Michaela FryeAbstract:Posttranscriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and are often organized by the chromatoid body. Here, we identify the RNA Methyltransferase NSun2 as a novel component of the chromatoid body and, further, show that NSun2 is essential for germ cell differentiation in the mouse testis. In NSun2-depleted testes, genes encoding Ddx4, Miwi, and Tudor domain-containing (Tdr) proteins are repressed, indicating that RNA-processing and posttranscriptional pathways are impaired. Loss of NSun2 specifically blocked meiotic progression of germ cells into the pachytene stage, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 and Dnmt2, the only other cytosine-5 RNA Methyltransferase characterized to date, indicating that Dnmt2 was not functionally redundant with NSun2 in spermatogonial stem cells or Sertoli cells. Specific NSun2- and Dnmt2-methylated tRNAs decreased in abundance when both Methyltransferases were deleted, suggesting that RNA methylation pathways play an essential role in male germ cell differentiation.
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mutation in nsun2 which encodes an RNA Methyltransferase causes autosomal recessive intellectual disability
American Journal of Human Genetics, 2012Co-Authors: Shobbir Hussain, Joana V Flores, Muzammil Ahmad Khan, Muhammad Rafiq, Abdul Noor, Verena Rupp, Akshita K Vincent, Roland Malli, Falak Sher KhanAbstract:Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32–p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a Methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA Methyltransferase in human neurocognitive development.
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the nucleolar RNA Methyltransferase misu nsun2 is required for mitotic spindle stability
Journal of Cell Biology, 2009Co-Authors: Shobbir Hussain, Agata Kurowski, Sandra Blanco Benavente, Elisabete Nascimento, Ilaria Dragoni, Astrid Gillich, Peter Humphreys, Michaeala FryeAbstract:Myc-induced SUN domain–containing protein (Misu or NSun2) is a nucleolar RNA Methyltransferase important for c-Myc–induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA–protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.
Ying Huang - One of the best experts on this subject based on the ideXlab platform.
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beyond a ribosomal RNA Methyltransferase the wider role of mraw in dna methylation motility and colonization in escherichia coli o157 h7
Frontiers in Microbiology, 2019Co-Authors: Xuefang Xu, Ying Huang, Heng Zhang, Yuan Zhang, Xiaoyuan Wang, Dai Wang, Changde Wu, Zhongqiu Teng, Xiaojing Peng, Ji PuAbstract:Abstract MraW is a 16S rRNA Methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes were confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions of promoters and coding regions of the four flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA Methyltransferase activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.
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beyond a ribosomal RNA Methyltransferase the wider role of mraw in dna methylation motility and colonization in escherichia coli o157 h7
bioRxiv, 2018Co-Authors: Xuefang Xu, Ying Huang, Heng Zhang, Yuan Zhang, Xiaoyuan Wang, Dai Wang, Ji Pu, Hongqing Zhao, Xuancheng Lu, Shuangshuang LuAbstract:MraW (RsmH) is an AdoMet-dependent 16S rRNA Methyltransferase conserved in bacteria and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus in host animals. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that deletion of mraW led to decreased motility and flagellar production. Whole-genome bisulfite sequencing showed genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant. The methylation level of 4 flagellar gene sequences were further confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated the transcription of these genes was also affected. MraW was observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common motif in differentially methylated regions of promoters and coding regions of the 4 flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA Methyltransferase (DNMT) activity assay in vitro. The mraW mutant colonized poorer than wild type in mice. we further found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and has a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.
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structural insights into mechanisms of the small RNA Methyltransferase hen1
Nature, 2009Co-Authors: Ying Huang, Lijuan Ji, Qichen Huang, Dmitry G Vassylyev, Xuemei ChenAbstract:Some of the small RNAs involved in RNA silencing require addition on their 3′ terminal nucleotide of a 2′-O-methyl group in order for the precursor RNA to be processed correctly. This modification is performed by the HEN1 RNA Methyltransferase, using AdoMet as a methyl donor. In this study, Ma and colleagues have solved the structure of a plant HEN1 in complex with an RNA duplex and the cofactor product, AdoHcy. The structure reveals how the enzyme recognizes the correct substrate and suggests a new mechanism for methylation. Some of the small RNAs involved in RNA silencing require the addition of a 2′-O-methyl group on the 3′ terminal nucleotide in order for the precursor RNA to be correctly processed. This modification is performed by the HEN1 RNA Methyltransferase, the crystal structure of which — from Arabidopsis — is now solved. RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ∼20–30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2′-O-methylation on the 3′ terminal nucleotide3,4,5,6. A conserved S-adenosyl-l-methionine-dependent RNA Methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3,4,5,7,8. Here we report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the Methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2′ and 3′ hydroxyls on the 3′-terminal nucleotide and four invariant residues in the active site of the Methyltransferase domain suggests a novel Mg2+-dependent 2′-O-methylation mechanism.
Robert M. Stroud - One of the best experts on this subject based on the ideXlab platform.
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redox reactions of the iron sulfur cluster in a ribosomal RNA Methyltransferase ruma optical and epr studies
Journal of Biological Chemistry, 2004Co-Authors: Sanjay Agarwalla, Robert M. Stroud, Betty J. GaffneyAbstract:Abstract An unprecedented [4Fe-4S] iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA (Agarwalla, S., Kealey, J. T., Santi, D. V., and Stroud, R. M. (2002) J. Biol. Chem. 277, 8835–8840; Lee, T. T., Agarwalla, S., and Stroud, R. M. (2004) Structure 12, 397–407). Methyltransferase reactions do not involve a redox step. To understand the structural and functional roles of the cluster in RumA, we have characterized redox reactions of the iron-sulfur cluster. As isolated aerobically, RumA exhibits a visible absorbance maximum at 390 nm and is EPR silent. It cannot be reduced by anaerobic additions of dithionite. Photoreduction by deazariboflavin/EDTA gives EPR spectra, the quantity (56% of S = 1/2 species) and details (gav ∼ 1.96–1.93) of which indicate a [4Fe-4S]1+ cluster in the reduced RumA. Oxidation of RumA by ferricyanide leads to loss of the 390-nm band and appearance of lower intensity bands at 444 and 520 nm. EPR spectra of ferricyanide-oxidized RumA show a fraction (<8%) of the FeS cluster trapped in the [3Fe-4S]1+ form (gav ∼ 2.011) together with unusual radical-like spectrum (g′ values 2.015, 2.00, and 1.95). RumA also reacts with nitric oxide to give EPR spectra characteristic of the protein-bound iron dinitrosyl species. Oxidation of the cluster leads to its decomposition and that could be a mechanism for regulating the activity of RumA under conditions of oxidative stress in the cell. Sequence data base searches revealed that RumA homologs are widespread in various kingdoms of life and contain a conserved and unique iron-sulfur cluster binding motif, CX5CGGC.
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Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA
2004Co-Authors: Sanjay Agarwalla, Robert M. Stroud, Betty J. GaffneyAbstract:An unprecedented [4Fe-4S] iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA (Agarwalla, S., Kealey, J. T., Santi, D. V., and Stroud, R. M. (2002) J. Biol. Chem. 277, 8835–8840; Lee, T. T., Agarwalla, S., and Stroud, R. M. (2004) Structure 12, 397–407). Methyltransferase reactions do not involve a redox step. To understand the structural and functional roles of the cluster in RumA, we have characterized redox reactions of the iron-sulfur cluster. As isolated aerobically, RumA exhibits a visible absorbance maximum at 390 nm and is EPR silent. It cannot be reduced by anaerobic additions of dithionite. Photoreduction by deazariboflavin/EDTA gives EPR spectra, the quantity (56% of S ! 1/2 species) and details (gav ! 1.96–1.93) of which indicate a [4Fe4S]1" cluster in the reduced RumA. Oxidation of RumA by ferricyanide leads to loss of the 390-nm band and appearance of lower intensity bands at 444 and 520 nm. EPR spectra of ferricyanide-oxidized RumA show a fraction (
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the first structure of an RNA m5c Methyltransferase fmu provides insight into catalytic mechanism and specific binding of RNA substrate
Structure, 2003Co-Authors: Paul G Foster, Christa R Nunes, Patricia J Greene, Demetri T Moustakas, Robert M. StroudAbstract:Abstract The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA Methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m 5 C Methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.
