Search results for "RNA modification"

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

2015

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pm…

2020

Abstract RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS s…

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…

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…

Kti12, a PSTK-like tRNA dependent ATPase essential for tRNA modification by Elongator

2019

Abstract Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure and function of its regulatory factor Kti12 have remained elusive. Here, we present the crystal structure of Kti12′s nucleotide hydrolase domain trapped in a transition state of ATP hydrolysis. The structure reveals striking similarities to an O-phosphoseryl-tRNA kinase involved in the selenocysteine pathway. Both proteins employ similar …

MODOMICS: a database of RNA modification pathways—2013 update

2012

MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, RNA-modifying enzymes and location of modified residues in RNA sequences. In the current database version, accessible at http://modomics.genesilico.pl, we included new features: a census of human and yeast snoRNAs involved in RNA-guided RNA modification, a new section covering the 5′-end capping process, and a catalogue of ‘building blocks’ for chemical synthesis of a large variety of modified nucleosides. The MODOMICS collections of RNA modifications, RNA-modifying enzymes and modified RNAs have been also updated. A…

Analysis of pseudouridines and other RNA modifications using hydraPsiSeq protocol

2021

Detection of RNA modified nucleotides using deep sequencing can be performed by several approaches, including antibody-driven enrichment and natural or chemically induced RT signatures. However, only very few RNA modified nucleotides generate natural RT signatures and antibody-driven enrichment heavily depends on the quality of antibodies used and may be highly biased. Thus, the use of chemically-induced RT signatures is now considered as the most trusted experimental approach. In addition, the use of chemical reagents allows inclusion of simple "mock-treated" controls, to exclude spontaneous RT arrests, SNPs and other misincorporation-prone sites. Hydrazine is a well-known RNA-specific rea…

Mapping and Quantification of tRNA 2′-O-Methylation by RiboMethSeq

2018

Current development of epitranscriptomics field requires efficient experimental protocols for precise mapping and quantification of various modified nucleotides in RNA. Despite important advances in the field during the last 10 years, this task is still extremely laborious and time-consuming, even when high-throughput analytical approaches are employed. Moreover, only a very limited subset of RNA modifications can be detected and only rarely be quantified by these powerful techniques. In the past, we developed and successfully applied alkaline fragmentation-based RiboMethSeq approach for mapping and precise quantification of multiple 2'-O-methylation residues in ribosomal RNA. Here we descr…

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…

Absolute quantification of noncoding RNA by microscale thermophoresis

2019

Abstract Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA‐Seq‐based quantification approaches, strongly suggesting a bias due to tRNA modifica…