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https://www.readbyqxmd.com/read/28730304/novel-ribonuclease-activity-of-cusativin-from-cucumis-sativus-for-mapping-nucleoside-modifications-in-rna
#1
Balasubrahmanyam Addepalli, Sarah Venus, Priti Thakur, Patrick A Limbach
A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein...
July 20, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28712493/crispr-cas9-enabled-multiplex-genome-editing-and-its-application
#2
Bastian Minkenberg, Matthew Wheatley, Yinong Yang
The CRISPR/Cas9 system is a prevalent and versatile genome-editing tool of choice for basic and applied biological research. An exchange of a 20-bp spacer sequence in the gRNA can easily reprogram Cas9 to target a different DNA site. By expressing or providing multiple gRNAs, the system also enables multiplex genome editing at high efficiencies. Current approaches for providing multiple gRNAs in vivo include the use of multigene cassettes to express several gRNAs, Csy4-based excision, arrays of crRNAs, ribozyme-flanked gRNAs, tRNA-dependent cleavage of gRNAs, and direct introduction of Cas9 proteins preloaded with different gRNAs...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/28684555/statistically-robust-methylation-calling-for-whole-transcriptome-bisulfite-sequencing-reveals-distinct-methylation-patterns-for-mouse-rnas
#3
Carine Legrand, Francesca Tuorto, Mark Hartmann, Reinhard Liebers, Dominik Jacob, Mark Helm, Frank Lyko
(Cytosine-5) RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA (cytosine-5) methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification...
July 6, 2017: Genome Research
https://www.readbyqxmd.com/read/28655838/nonredox-thiolation-in-trna-occurring-via-sulfur-activation-by-a-4fe-4s-cluster
#4
Simon Arragain, Ornella Bimai, Pierre Legrand, Sylvain Caillat, Jean-Luc Ravanat, Nadia Touati, Laurent Binet, Mohamed Atta, Marc Fontecave, Béatrice Golinelli-Pimpaneau
Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity...
July 11, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28643349/a-qm-mm-study-of-the-catalytic-mechanism-of-sam-methyltransferase-rlmn-from-escherichia-coli
#5
Chenxiao Zhao, Lihua Dong, Yongjun Liu
RlmN is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C2 methylation of adenosine 2503 (A2503) in 23S rRNA and adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNA). The catalytic reaction of RlmN is distinctly different from that of typical SAM-dependent methyltransferases that employs an SN 2 mechanism, but follows a ping-pong mechanism which involves the intermediate methylation of a conserved cysteine residue. Recently, the x-ray structure of a key intermediate in the RlmN reaction has been reported, allowing us to perform combined quantum mechanics and molecular mechanics (QM/MM) calculations to delineate the reaction details of RlmN at atomic level...
June 23, 2017: Proteins
https://www.readbyqxmd.com/read/28607080/structural-studies-of-viperin-an-antiviral-radical-sam-enzyme
#6
Michael K Fenwick, Yue Li, Peter Cresswell, Yorgo Modis, Steven E Ealick
Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure...
June 27, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28601227/trmd-a-methyl-transferase-for-trna-methylation-with-m-1-g37
#7
Ya-Ming Hou, Ryuma Matsubara, Ryuichi Takase, Isao Masuda, Joanna I Sulkowska
TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated m(1)G37 in tRNA. TrmD is specific to and essential for bacterial growth, and it is fundamentally distinct from its eukaryotic and archaeal counterpart Trm5. TrmD is unusual by using a topological protein knot to bind AdoMet. Despite its restricted mobility, the TrmD knot has complex dynamics necessary to transmit the signal of AdoMet binding to promote tRNA binding and methyl transfer. Mutations in the TrmD knot block this intramolecular signaling and decrease the synthesis of m(1)G37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis...
2017: Enzymes
https://www.readbyqxmd.com/read/28593035/smchd1-regulates-a-limited-set-of-gene-clusters-on-autosomal-chromosomes
#8
Amanda G Mason, Roderick C Slieker, Judit Balog, Richard J L F Lemmers, Chao-Jen Wong, Zizhen Yao, Jong-Won Lim, Galina N Filippova, Enrico Ne, Rabi Tawil, Bas T Heijmans, Stephen J Tapscott, Silvère M van der Maarel
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance...
2017: Skeletal Muscle
https://www.readbyqxmd.com/read/28587678/smchd1-regulates-a-limited-set-of-gene-clusters-on-autosomal-chromosomes
#9
Amanda G Mason, Roderick C Slieker, Judit Balog, Richard J L F Lemmers, Chao-Jen Wong, Zizhen Yao, Jong-Won Lim, Galina N Filippova, Enrico Ne, Rabi Tawil, Bas T Heijmans, Stephen J Tapscott, Silvère M van der Maarel
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance...
June 6, 2017: Skeletal Muscle
https://www.readbyqxmd.com/read/28576863/evolving-mistranslating-trnas-through-a-phenotypically-ambivalent-intermediate-in-saccharomyces-cerevisiae
#10
Matthew D Berg, Kyle S Hoffman, Julie Genereaux, Safee Mian, Ryan S Trussler, David B Haniford, Patrick O'Donoghue, Christopher J Brandl
The genetic code converts information from nucleic acid into protein. The genetic code was thought to be immutable, yet many examples in nature indicate that variations to the code provide a selective advantage. We used a sensitive selection system involving suppression of a deleterious allele (tti2-L187P) in Saccharomyces cerevisiae to detect mistranslation and identify mechanisms that allow genetic code evolution. Though tRNA(Ser) containing a proline anticodon (UGG) is toxic, using our selection system, we identified four tRNA(Ser)UGG variants, each with a single mutation, that mistranslate at a tolerable level...
June 2, 2017: Genetics
https://www.readbyqxmd.com/read/28576826/archaeal-fibrillarin-nop5-heterodimer-2-o-methylates-rna-independently-of-the-c-d-guide-rnp-particle
#11
Miglė Tomkuvienė, Janina Ličytė, Ingrida Olendraitė, Zita Liutkevičiūtė, Béatrice Clouet-d'Orval, Saulius Klimašauskas
Archaeal fibrillarin (aFib) is a well-characterized S-Adenosyl methionine (SAM)-dependent RNA 2'-O-methyltransferase that is known to act in a large C/D ribonucleoprotein (RNP) complex together with Nop5 and L7Ae proteins and a box C/D guide RNA. In the reaction, the guide RNA is critical to direct the methylation reaction to a specific site in tRNA or rRNA by sequence complementarity. Here we show that Pyrococcus abyssi aFib-Nop5 heterodimer can alone perform a SAM-dependent 2'-O-methylation of 16S and 23S ribosomal RNAs in vitro independently of L7Ae and C/D guide RNAs...
June 2, 2017: RNA
https://www.readbyqxmd.com/read/28575027/linking-epigenetic-function-to-electrostatics-the-dnmt2-structural-model-example
#12
Gilberto Cavalheiro Vieira, Gustavo Fioravanti Vieira, Marialva Sinigaglia, Vera Lúcia da Silva Valente
The amino acid sequence of DNMT2 is very similar to the catalytic domains of bacterial and eukaryotic proteins. However, there is great variability in the region of recognition of the target sequence. While bacterial DNMT2 acts as a DNA methyltransferase, previous studies have indicated low DNA methylation activity in eukaryotic DNMT2, with preference by tRNA methylation. Drosophilids are known as DNMT2-only species and the DNA methylation phenomenon is a not elucidated case yet, as well as the ontogenetic and physiologic importance of DNMT2 for this species group...
2017: PloS One
https://www.readbyqxmd.com/read/28567122/a-functional-role-for-the-monomethylated-gln-51-and-lys-53-residues-of-the-49ggqtk53-motif-of-el42-from-human-80s-ribosomes
#13
Stéphanie Eustache, Jean-Bernard Créchet, Tahar Bouceba, Jun-Ichi Nakayama, Mayo Tanaka, Mieko Suzuki, Anne Woisard, Pierre Tuffery, Soria Baouz, Codjo Hountondji
BACKGROUND: We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA...
2017: Open Biochemistry Journal
https://www.readbyqxmd.com/read/28544464/structural-and-biochemical-insights-into-the-2-o-methylation-of-pyrimidines-34-in-trna
#14
Panjiao Pang, Xiangyu Deng, Zhong Wang, Wei Xie
tRNA molecules undergo extensive modifications during their maturation and these modifications play important cellular roles. TrmL is a tRNA-modification enzyme that catalyzes the transfer of a methyl group to the 2'-hydroxyl group of the pyrimidines at the wobble position 34 in two tRNA(L)(eu) isoacceptors, but the mechanism remains elusive. In this study, we determined the crystal structure of TrmL from Thermus thermophilus (TtTrmL) to 1.7 Å. The enzyme contains only the conserved minimal SPOUT fold, but displays distinct biochemical behavior from its Escherichia coli counterpart, EcTrmL...
May 25, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28531330/structural-basis-for-substrate-binding-and-catalytic-mechanism-of-a-human-rna-m5c-methyltransferase-nsun6
#15
Ru-Juan Liu, Tao Long, Jing Li, Hao Li, En-Duo Wang
5-methylcytosine (m5C) modifications of RNA are ubiquitous in nature and play important roles in many biological processes such as protein translational regulation, RNA processing and stress response. Aberrant expressions of RNA:m5C methyltransferases are closely associated with various human diseases including cancers. However, no structural information for RNA-bound RNA:m5C methyltransferase was available until now, hindering elucidation of the catalytic mechanism behind RNA:m5C methylation. Here, we have solved the structures of NSun6, a human tRNA:m5C methyltransferase, in the apo form and in complex with a full-length tRNA substrate...
May 22, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28506829/an-asymmetric-dimeric-structure-of-trmj-trna-methyltransferase-from-zymomonas-mobilis-with-a-flexible-c-terminal-dimer
#16
Do-Heon Gu, Mi-Young Park, Jeong-Sun Kim
The tRNA methyltransferase J (TrmJ) and D (TrmD) catalyze the transferring reaction of a methyl group to the tRNA anticodon loop. They commonly have the N-terminal domain (NTD) and the C-terminal domain (CTD). Whereas two monomeric CTDs symmetrically interact with a dimeric NTD in TrmD, a CTD dimer has exhibited an asymmetric interaction with the NTD dimer in the presence of a product. The elucidated apo-structure of the full-length TrmJ from Zymomonas mobilis ZM4 shows a dimeric CTD that asymmetrically interacts with the NTD dimer, thereby distributing non-symmetrical potential charge on the both side of the protein surface...
May 12, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28488916/observing-the-fate-of-trna-and-its-modifications-by-nucleic-acid-isotope-labeling-mass-spectrometry-nail-ms
#17
Matthias Heiss, Valentin F Reichle, Stefanie Kellner
RNA in yeast, especially rRNA and tRNA are heavily modified to fulfill their function in protein translation. Using biosynthetic stable isotope labeled internal standards we quantified 12 modified nucleosides in tRNA from S. cerevisiae over 24 hours. We observed different quantities of modified nucleosides in dependence of the growth phase. To elucidate the underlying mechanism of the observed tRNA modification profile adaptation, it is necessary to distinguish the pre-existing tRNA pool and its modifications from newly-synthesized tRNAs...
May 10, 2017: RNA Biology
https://www.readbyqxmd.com/read/28476776/cytosine-methylation-by-dnmt2-facilitates-stability-and-survival-of-hiv-1-rna-in-the-host-cell-during-infection
#18
Rachana Roshan Dev, Rakesh Ganji, Satya Prakash Singh, Sundarasamy Mahalingam, Sharmistha Banerjee, Sanjeev Khosla
The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner...
June 6, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28418649/nature-s-selection-of-geranyl-group-as-a-trna-modification-the-effects-of-chain-length-on-base-pairing-specificity
#19
Phensinee Haruehanroengra, Sweta Vangaveti, Srivathsan V Ranganathan, Rui Wang, Alan Chen, Jia Sheng
The recently discovered geranyl modification on the 2-thio position of wobble U34 residues in tRNA(Glu), tRNA(Lys), and tRNA(Gln) in several bacteria has been found to enhance the U:G pairing specificity and reduce the frameshifting error during translation. It is a fundamentally interesting question why nature chose a C10 terpene group in tRNA systems. In this study, we explore the significance of the terpene length on base-paring stability and specificity using a series of 2-thiouridine analogues containing different lengths of carbon chains, namely, methyl- (C1), dimethylallyl- (C5), and farnesyl-modified (C15) 2-thiothymidines in a DNA duplex...
April 18, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28371071/selective-enzymatic-demethylation-of-n-2-n-2-dimethylguanosine-in-rna-and-its-application-in-high-throughput-trna-sequencing
#20
Qing Dai, Guanqun Zheng, Michael H Schwartz, Wesley C Clark, Tao Pan
The abundant Watson-Crick face methylations in biological RNAs such as N(1) -methyladenosine (m(1) A), N(1) -methylguanosine (m(1) G), N(3) -methylcytosine (m(3) C), and N(2) ,N(2) -dimethylguanosine (m(2)2 G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m(1) A, m(1) G, m(3) C modifications in transfer RNA (tRNA), but they work poorly on m(2)2 G...
April 24, 2017: Angewandte Chemie
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