Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field.

A role for β-1,6- and β-1,3-glucans in kinetochore function in Saccharomyces cerevisiae.

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A, and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for β-1,6- and β-1,3-glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae.

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing.

Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis.

A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine.

More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis.

Methylation of CENP-A/Cse4 on arginine 143 and lysine 131 regulates kinetochore stability in yeast.

Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome.

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit.

Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification.

Queuosine salvage in fission yeast by Qng1-mediated hydrolysis to queuine.

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside that is found at position 34, also known as the wobble position, of tRNAs with a GUN anticodon, and Q ensures faithful translation of the respective C- and U-ending codons. While Q is present in tRNAs in most eukaryotes, only bacteria can synthesize it denovo. In contrast, eukaryotes rely on external sources like their food and the gut microbiome in order to Q-modify their tRNAs, and Q therefore can be regarded as a micronutrient.

Increased CG, CHG and CHH methylation at the cycloidea gene in the "Peloria" mutant of Linaria vulgaris.

Heritable DNA methylation variation is frequently observed in natural populations of plants, but is thought mostly to be functionally inconsequential. An exception to this is the "Peloria" mutant of Linaria vulgaris, which was originally described by Carl von Linné in 1744. A study in 1999 found that the Peloria phenotype is caused by an epiallele of the L.

Dynamics of SAS-I mediated H4 K16 acetylation during DNA replication in yeast.

The acetylation of H4 lysine 16 (H4 K16Ac) in Saccharomyces cerevisiae counteracts the binding of the heterochromatin complex SIR to chromatin and inhibits gene silencing. Contrary to other histone acetylation marks, the H4 K16Ac level is high on genes with low transcription, whereas highly transcribed genes show low H4 K16Ac. Approximately 60% of cellular H4 K16Ac in S.

The ATAD2/ANCCA homolog Yta7 cooperates with Scm3 to deposit Cse4 at the centromere in yeast.

The AAA ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the homolog Yta7 is a deposition factor for the centromeric histone H3 variant Cse4 at the centromere in yeast. Yta7 regulates the levels of centromeric Cse4 in that causes reduced Cse4 deposition, whereas overexpression causes increased Cse4 deposition.

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes, function to connect centromeric chromatin to microtubules of the mitotic spindle. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules.

Queuine links translational control in eukaryotes to a micronutrient from bacteria.

In eukaryotes, the wobble position of tRNA with a GUN anticodon is modified to the 7-deaza-guanosine derivative queuosine (Q34), but the original source of Q is bacterial, since Q is synthesized by eubacteria and salvaged by eukaryotes for incorporation into tRNA. Q34 modification stimulates Dnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodon loop in Schizosaccharomyces pombe. Here, we show by ribosome profiling in S.

Division of labour: tRNA methylation by the NSun2 tRNA methyltransferases Trm4a and Trm4b in fission yeast.

Enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. In contrast to most other organisms, fission yeast Schizosaccharomyces pombe carries two Trm4/NSun2 homologs, Trm4a (SPAC17D4.04) and Trm4b (SPAC23C4.

The kinetochore module Okp1/Ame1 is a reader for N-terminal modifications on the centromeric histone Cse4.

Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from regulate molecular interactions at the inner kinetochore.

Queuosine-modified tRNAs confer nutritional control of protein translation.

Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q-tRNA levels promote Dnmt2-mediated methylation of tRNA Asp and control translational speed of Q-decoded codons as well as at near-cognate codons.

A role for CENP-A/Cse4 phosphorylation on serine 33 in deposition at the centromere.

Centromeres are the sites of assembly of the kinetochore, which connect the chromatids to the microtubules for sister chromatid segregation during cell division. Centromeres are characterized by the presence of the histone H3 variant CENP-A (termed Cse4 in Saccharomyces cerevisiae). Here, we investigated the function of serine 33 phosphorylation of Cse4 (Cse4-S33ph) in S.

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification.

Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA) methyltransferases for cytosine-5 methylation, foremost C38 (m5C38) of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe.

Variants of the Sir4 Coiled-Coil Domain Improve Binding to Sir3 for Heterochromatin Formation in .

Heterochromatin formation in the yeast is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing function , as well binding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type loci and to a Sir3 version with a mutation in this loop.

Defects in methylation of arginine 37 on CENP-A/Cse4 are compensated by the ubiquitin ligase complex Ubr2/Mub1.

The kinetochore, a supramolecular protein complex, provides the physical connection between chromatin and the microtubule and ensures correct chromosome segregation during mitosis. Centromeric regions are marked by the presence of the histone H3 variant CENP-A. Cse4, the CENP-A homologue from Saccharomyces cerevisiae, is methylated on arginine 37 in its N-terminus (R37), and the absence of methylation (cse4-R37A) causes synthetic genetic defects in combination with mutations or deletions in genes encoding components of the Ctf19/CCAN complex and with the CDEI binding protein Cbf1.

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation.

A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research.

Widespread colocalization of the Drosophila histone acetyltransferase homolog MYST5 with DREF and insulator proteins at active genes.

MYST family histone acetyltransferases play important roles in gene regulation. Here, we have characterized the Drosophila MYST histone acetyltransferase (HAT) encoded by cg1894, whose closest homolog is Drosophila MOF, and which we have termed MYST5. We found it localized to a large number of interbands as well as to the telomeres of polytene chromosomes, and it showed strong colocalization with the interband protein Z4/Putzig and RNA polymerase II.

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

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.

A link between Sas2-mediated H4 K16 acetylation, chromatin assembly in S-phase by CAF-I and Asf1, and nucleosome assembly by Spt6 during transcription.

The histone acetyltransferase Sas2 is part of the SAS-I complex and acetylates lysine 16 of histone H4 (H4 K16Ac) in the genome of Saccharomyces cerevisiae. Sas2-mediated H4 K16Ac is strongest over the coding region of genes with low expression. However, it is unclear how Sas2-mediated acetylation is incorporated into chromatin.

A method for high-throughput analysis of chronological aging in Schizosaccharomyces pombe.

The measurement of chronological life span (CLS) in Schizosaccharomyces pombe is traditionally performed by plating back aliquots of aging liquid cultures on solid medium and counting the number of colony-forming units (CFU). However, this method is labor and cost intensive and therefore not amenable to high-throughput screening. Here, we describe a simple method for CLS measurement using aging minicultures in microtiter plates and batch plate-back for the determination of culture viability.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice.