0000000001309842
AUTHOR
Stefanie Kellner
showing 14 related works from this author
Related haloarchaeal pleomorphic viruses contain different genome types
2012
Archaeal viruses have been the subject of recent interest due to the diversity discovered in their virion architectures. Recently, a new group of haloarchaeal pleomorphic viruses has been discovered. It is distinctive in terms of the virion morphology and different genome types (ssDNA/dsDNA) harboured by rather closely related representatives. To date there are seven isolated viruses belonging to this group. Most of these share a cluster of five conserved genes, two of which encode major structural proteins. Putative proviruses and proviral remnants containing homologues of the conserved gene cluster were also identified suggesting a long-standing relationship of these viruses with their ho…
The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA
2016
The amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNAAsp, we showed that this natural Dnmt2 substrate could be engineered to employ R…
Variable presence of 5-methylcytosine in commercial RNA and DNA
2015
Nucleoside methylations and other nucleic acid modifications have recently encountered a surge in interest, prompted, among other things, by the detection of methylation and active demethylation of DNA and mRNA by similar mechanisms. In DNA, deoxycytidine methylation by Dnmt enzymes generates 5-methyldeoxycytidine,1 an important epigenetic mark that typically causes inactivation of transcription of the methylated promoter region. Recent exciting developments have shown that these marks are not concrete-cast, but can be actively removed by the oxidative action of TET enzymes,2 which generate, through a series of 2-electron oxidations, first hydroxymethylcytidine (hm5C), then formyldeoxycytid…
Partial Methylation at Am100 in 18S rRNA of Baker's Yeast Reveals Ribosome Heterogeneity on the Level of Eukaryotic rRNA Modification
2014
Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in t…
RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis.
2012
The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylate…
A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA
2011
A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as dem…
Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders.
2014
Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5' tRNA-derived small RNA fragments. Accumulation of 5' tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell siz…
Detection of RNA modifications
2010
RNA nucleotide modifications are typically of low abundance and frequently go unnoticed by standard detection methods of molecular biology and cell biology. With a burst of knowledge intruding from such diverse areas as genomics, structural biology, regulation of gene expression and immunology, it becomes increasingly clear that many exciting functions of nucleotide modifications remain to be explored. It follows in turn that the biology of nucleotide modification and editing is a field poised to rapidly gain importance in a variety of fields. The detection and analysis of nucleotide modifications present a clear limitation in this respect. Here, various methods for detection of nucleotide …
Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs
2019
AbstracttRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein conta…
Structure-Function Relationship of Substituted Bromomethylcoumarins in Nucleoside Specificity of RNA Alkylation
2013
Selective alkylation of RNA nucleotides is an important field of RNA biochemistry, e.g. in applications of fluorescent labeling or in structural probing experiments, yet detailed structure-function studies of labeling agents are rare. Here, bromomethylcoumarins as reactive compounds for fluorescent labeling of RNA are developed as an attractive scaffold on which electronic properties can be modulated by varying the substituents. Six different 4-bromomethyl-coumarins of various substitution patterns were tested for nucleotide specificity of RNA alkylation using tRNA from Escherichia coli as substrate. Using semi-quantitative LC-MS/MS analysis, reactions at mildly acidic and slightly alkaline…
Profiling of RNA modifications by multiplexed stable isotope labelling
2014
The combination of (15)N/(13)C stable isotope labelling (SIL) and LC-MS/MS revealed a total of 52 modifications in RNA from E. coli and yeast, including 10 previously undescribed modifications. Two modifications, N-ribosylnicotinamide and 2-methylthioadenosine, were newly detected in species hitherto thought not to contain these modifications.
Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120.
2015
International audience; Modified nucleotide 5-methylcytosine (m(5)C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA: m(5)C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m(5)C-MTase activity, which restores m(5)C formation at position 2870 in domain V of 25S rRNA in a nop2 Delta …
Absolute and relative quantification of RNA modifications via biosynthetic isotopomers
2014
In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC-MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding (13)C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single mod…
Variable presence of 5-methylcytosine in commercial RNA and DNA
2015
5-methylcytosine (m5C, mC) is a naturally occurring nucleoside modification in both RNA and DNA. Its presence in DNA is a widely accepted epigenetic mark for transcription inactivation. In RNA, its appearance in different coding as well as non-coding RNA implies multiple functions, with regulation of gene expression as a common denominator. Here we report on the serendipitous discovery of m5C in synthetic oligonucleotides, which prompted a systematic quantification in synthetic DNA and RNA of academic as well as of commercial origin. For both types of oligonucleotides, m5C was identified by comparison of fragmentation pattern and retention time with authentic standards by highly sensitive L…