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Cloning and characterization of PIMT, a protein with a methyltransferase domain, which interacts with and enhances nuclear receptor coactivator PRIP function

Zhu, Yijun; Qi, Chao; Cao, Wen-Qing; Yeldandi, Anjana V.; Rao, M. Sambasiva; Reddy, Janardan K.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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The nuclear receptor coactivators participate in the transcriptional activation of specific genes by nuclear receptors. In this study, we report the isolation of a nuclear receptor coactivator-interacting protein from a human liver cDNA library by using the coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) (ASC2/AIB3/RAP250/NRC/TRBP) as bait in a yeast two-hybrid screen. Human PRIP-interacting protein cDNA has an ORF of 2,556 nucleotides, encodes a protein with 852 amino acids, and contains a 9-aa VVDAFCGVG methyltransferase motif I and an invariant GXXGXXI segment found in K-homology motifs of many RNA-binding proteins. The gene encoding this protein, designated PRIP-interacting protein with methyltransferase domain (PIMT), is localized on chromosome 8q11 and spans more than 40 kb. PIMT mRNA is ubiquitously expressed, with a high level of expression in heart, skeletal muscle, kidney, liver, and placenta. Using the immunofluorescence localization method, we found that PIMT and PRIP proteins appear colocalized in the nucleus. PIMT strongly interacts with PRIP under in vitro and in vivo conditions, and the PIMT-binding site on PRIP is in the region encompassing amino acids 773–927. PIMT binds S-adenosyl-l-methionine...

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

HALL, MEGAN P.; HO, C. KIONG
Fonte: Copyright 2006 by RNA Society Publicador: Copyright 2006 by RNA Society
Tipo: Artigo de Revista Científica
Publicado em /03/2006 Português
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The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m7GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324–amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m7GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predicted—on the basis of the crystal structure of Ecm1—to be located at or near the active site identified six conserved residues in the putative AdoMet- or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050–amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain...

Preparation, crystallization and preliminary X-ray diffraction analysis of PH1948, predicted RNA methyltransferase from Pyrococcus horikoshii *

Gao, Yong-gui; Yao, Min; Tanaka, Isao
Fonte: Zhejiang University Press Publicador: Zhejiang University Press
Tipo: Artigo de Revista Científica
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RNA methyltransferase is responsible for transferring methyl and resulting in methylation on the bases or ribose ring of RNA, which existed widely but mostly remains an open question. A recombinant protein PH1948 predicting RNA methyltransferase from Pyrococcus horikoshii OT3 has been crystallized. The crystals of selenomethionyl PH1948 belong to space group C2, with unit-cell parameters a=207.0 Å, b=43.1 Å, c=118.2 Å, β=92.1°, and diffract X-rays to 2.2 Å resolution. The V M value was determined to be 2.8 Å3/Da, indicating the presence of four protein molecules in the asymmetric unit.

Aurora-B Regulates RNA Methyltransferase NSUN2

Sakita-Suto, Shiho; Kanda, Akifumi; Suzuki, Fumio; Sato, Sunao; Takata, Takashi; Tatsuka, Masaaki
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /03/2007 Português
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Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G1 phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.

New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

Nield, Blair S.; Willows, Robert D.; Torda, Andrew E.; Gillings, Michael R.; Holmes, Andrew J.; Nevalainen, K.M. Helena; Stokes, H.W.; Mabbutt, Bridget C.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /06/2004 Português
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By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%–28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample...

Subcellular Localization and RNA Interference of an RNA Methyltransferase Gene from Silkworm, Bombyx Mori

Nie, Zuoming; Zhou, Ruobing; Chen, Jian; Wang, Dan; Lv, Zhengbing; He, Pingan; Wang, Xuedong; Shen, Hongdan; Wu, Xiangfu; Zhang, Yaozhou
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
Português
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RNA methylation, which is a form of posttranscriptional modification, is catalyzed by S-adenosyl-L-methionone-dependent RNA methyltransterases (RNA MTases). We have identified a novel silkworm gene, BmRNAMTase, containing a 369-bp open reading frame that encodes a putative protein containing 122 amino acid residues and having a molecular weight of 13.88 kd. We expressed a recombinant His-tagged BmRNAMTase in E. coli BL21 (DE3), purified the fusion protein by metal-chelation affinity chromatography, and injected a New Zealand rabbit with the purified protein to generate anti-BmRNAMTase polyclonal antibodies. Immunohistochemistry revealed that BmRNAMTase is abundant in the cytoplasm of Bm5 cells. In addition, using RNA interference to reduce the intracellular activity and content of BmRNAMTase, we determined that this cytoplasmic RNA methyltransferase may be involved in preventing cell death in the silkworm.

Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism

Jurkowski, Tomasz P.; Meusburger, Madeleine; Phalke, Sameer; Helm, Mark; Nellen, Wolfgang; Reuter, Gunter; Jeltsch, Albert
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /08/2008 Português
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Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNAAsp. We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNAAsp confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

Scintillation proximity assay for measurement of RNA methylation

Baker, Matthew R.; Zarubica, Tamara; Wright, H. Tonie; Rife, Jason P.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA–protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC’. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC’ most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA–protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format.

The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability

Hussain, Shobbir; Benavente, Sandra Blanco; Nascimento, Elisabete; Dragoni, Ilaria; Kurowski, Agata; Gillich, Astrid; Humphreys, Peter; Frye, Michaela
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 13/07/2009 Português
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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.

Kinetic and functional analysis of the small RNA methyltransferase HEN1: The catalytic domain is essential for preferential modification of duplex RNA

Vilkaitis, Giedrius; Plotnikova, Alexandra; Klimašauskas, Saulius
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /10/2010 Português
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The HEN1 RNA methyltransferase from Arabidopsis thaliana catalyzes S-adenosyl-L-methionine (AdoMet)-dependent 2′-O-methylation at the 3′-termini of small double-stranded RNAs and is a crucial factor in the biogenesis of plant small noncoding RNAs, such as miRNAs or siRNAs. We performed functional and kinetic studies of the full-length HEN1 methyltransferase and its truncated form comprising the C-terminal part of the protein (residues 666–942) with a variety of model RNA substrates. Kinetic parameters obtained with natural RNA substrates indicate that HEN1 is highly catalytically efficient in the absence of any supplementary proteins. We find that the enzyme modifies individual strands in succession leading to complete methylation of an RNA duplex. The rates of methyl group transfer to individual strands of hemimethylated substrates under single turnover conditions are comparable with the multiple turnover rate under steady-state conditions, suggesting that release of reaction products is not a rate-limiting event in the reaction cycle. The truncated protein, which includes conserved motifs characteristic for AdoMet binding, efficiently modifies double-stranded RNA substrates in vitro; however, in contrast to the full-length methyltransferase...

Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

Jain, Ruchi; Shuman, Stewart
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /03/2011 Português
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The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganese-dependent RNA ribose 2′O-methyltransferase that marks the 3′ terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 3′ dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12–24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 3′ terminal ribonucleotide...

The RNA–Methyltransferase Misu (NSun2) Poises Epidermal Stem Cells to Differentiate

Blanco, Sandra; Kurowski, Agata; Nichols, Jennifer; Watt, Fiona M.; Benitah, Salvador Aznar; Frye, Michaela
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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46.29%
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.

Mutation in NSUN2, which Encodes an RNA Methyltransferase, Causes Autosomal-Recessive Intellectual Disability

Khan, Muzammil Ahmad; Rafiq, Muhammad Arshad; Noor, Abdul; Hussain, Shobbir; Flores, Joana V.; Rupp, Verena; Vincent, Akshita K.; Malli, Roland; Ali, Ghazanfar; Khan, Falak Sher; Ishak, Gisele E.; Doherty, Dan; Weksberg, Rosanna; Ayub, Muhammad; Win
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em 04/05/2012 Português
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46.23%
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...

The Mouse Cytosine-5 RNA Methyltransferase NSun2 Is a Component of the Chromatoid Body and Required for Testis Differentiation

Hussain, Shobbir; Tuorto, Francesca; Menon, Suraj; Blanco, Sandra; Cox, Claire; Flores, Joana V.; Watt, Stephen; Kudo, Nobuaki R.; Lyko, Frank; Frye, Michaela
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2013 Português
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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.

The Bin3 RNA methyltransferase (MePCE) targets 7SK RNA to control transcription and translation

Cosgrove, Michael; Ding, Ye; Rennie, William A.; Lane, Michael J.; Hanes, Steven D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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46.32%
Bin3 is a conserved RNA methyltransferase found in eukaryotes ranging from fission yeast to humans. It was originally discovered as a Bicoid-interacting protein in Drosophila, where it is required for anterior-posterior and dorso-ventral axis determination in the early embryo. The mammalian ortholog of Bin3 (BCDIN3), also known as methyl phosphate capping enzyme (MePCE), plays a key role in repressing transcription. In transcription, MePCE binds the non-coding 7SK RNA, which forms a scaffold for an RNA-protein complex that inhibits P-TEFb, an RNA polymerase II elongation factor. MePCE uses S-adenosyl methionine to transfer a methyl group onto the γ-phosphate of the 5′ guanosine of 7SK RNA generating an unusual cap structure that protects 7SK RNA from degradation. Bin3/MePCE also has a role in translation regulation. Initial studies in Drosophila indicate that Bin3 targets 7SK RNA and stabilizes a distinct RNA-protein complex that assembles on the 3′ UTR of caudal mRNAs to prevent translation initiation. Much remains to be learned about Bin3/MeCPE function, including how it recognizes 7SK RNA, what other RNA substrates it might target, and how widespread a role it plays in gene regulation and embryonic development.

Human RNA methyltransferase BCDIN3D regulates microRNA processing

Xhemalce, Blerta; Robson, Samuel C; Kouzarides, Tony
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 12/10/2012 Português
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46.13%
MicroRNAs regulate key biological processes and their aberrant expression may lead to cancer. The primary transcript of canonical miRNAs is sequentially cleaved by the RNase III enzymes, Drosha and Dicer, which generate 5′mono-phosphate ends that are important for subsequent miRNA functions. In particular, the recognition of the 5′mono-phosphate of pre-miRNAs by Dicer is important for precise and effective biogenesis of miRNAs. Here we identify a RNA-methyltransferase, BCDIN3D, that O-methylates this 5′mono-phosphate and negatively regulates miRNA maturation. Specifically, we show that BCDIN3D phospho-dimethylates pre-miR-145 both in vitro and in vivo and that phospho-dimethylated pre-miR-145 displays reduced processing by Dicer in vitro. Consistently, BCDIN3D depletion leads to lower pre-miR-145 and concomitantly increased mature miR-145 levels in breast cancer cells, which suppresses their tumorigenic phenotypes. Together, our results uncover a miRNA methylation pathway potentially involved in cancer that antagonizes the Dicer–dependent processing of miR-145 as well as other miRNAs.

Identification of a novel methyltransferase, Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in Saccharomyces cerevisiae

Sharma, Sunny; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified Rrp8 as a methyltransferase involved in m1A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m1A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase–HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-l-methionine–binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m1A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m1A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.

Discovery of the β-barrel–type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs

Kimura, Satoshi; Miyauchi, Kenjyo; Ikeuchi, Yoshiho; Thiaville, Patrick C.; de Crécy-Lagard, Valérie; Suzuki, Tsutomu
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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46.23%
Methylation is a versatile reaction involved in the synthesis and modification of biologically active molecules, including RNAs. N6-methyl-threonylcarbamoyl adenosine (m6t6A) is a post-transcriptional modification found at position 37 of tRNAs from bacteria, insect, plants, and mammals. Here, we report that in Escherichia coli, yaeB (renamed as trmO) encodes a tRNA methyltransferase responsible for the N6-methyl group of m6t6A in tRNAThr specific for ACY codons. TrmO has a unique single-sheeted β-barrel structure and does not belong to any known classes of methyltransferases. Recombinant TrmO employs S-adenosyl-L-methionine (AdoMet) as a methyl donor to methylate t6A to form m6t6A in tRNAThr. Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m6t6A was converted to cyclic t6A (ct6A), suggesting that t6A is a common precursor for both m6t6A and ct6A. Furthermore, N6-methylation of t6A enhanced the attenuation activity of the thr operon, suggesting that TrmO ensures efficient decoding of ACY. We also identified a human homolog, TRMO, indicating that m6t6A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

Haag, Sara; Warda, Ahmed S.; Kretschmer, Jens; Günnigmann, Manuel A.; Höbartner, Claudia; Bohnsack, Markus T.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /09/2015 Português
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Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m5C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m5C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNACys and tRNAThr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.

Structure of the Thiostrepton Resistance Methyltransferase·S-Adenosyl-l-methionine Complex and Its Interaction with Ribosomal RNA*

Dunstan, Mark S.; Hang, Pei C.; Zelinskaya, Natalia V.; Honek, John F.; Conn, Graeme L.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.