Biology subjects

Kellner, S.

Publications and source records attributed to Kellner, S..

9 recordsLinked to original sources

bioRxiv2025

Loss of characteristic species across German federal states detected by repeated mapping of protected habitats

Identifying the winners and losers of biodiversity change within different habitat types requires systematic monitoring. While such data are still lacking in Germany, species trends could be derived from previously untapped sources. Here, we derive temporal trends in plant species from data of repeated habitat mapping programs of three German states from 1977-2021, both across all habitat types per state and within habitat types. Consistently negative trends were found across all states for species preferring heaths and semi-natural grasslands, moist to wet grasslands, and coastal and marine habitats, including many endangered species. Consistently positive trends were found for species preferring scrubs, copses and field hedges, and for non-native species. Trends within habitat types showed negative trends for species characteristic of those habitat types. While trends varied among states, the overall patterns were very similar. This points to ongoing habitat degradation and common drivers of biodiversity change in Germany.

ecology↗

bioRxiv2024

Synergistic effects of tRNA modification defects in Escherichia coli K12.

Transfer RNAs (tRNAs) are essential components of the translation machinery and carry numerous post-transcriptional modifications that contribute to decoding accuracy, efficiency and cellular fitness. In Escherichia coli K-12, all tRNA modification pathways have been identified, yet the functional interactions between these pathways remain largely unexplored. Here, we systematically analyze genetic interactions between 29 non-essential tRNA modification genes using a pairwise synthetic lethal screen based on P1 transduction. Most combinations of tRNA modification gene deletions are tolerated during growth in rich medium; however, we identify five synthetically lethal pairs and fifteen additional combinations that display negative genetic interactions. Deletions of truA, which encodes the pseudouridine synthase responsible for modifications at positions 38-40 of multiple tRNAs, show the highest frequency of negative epistasis. Synthetic lethality associated with truA can be complemented by expression of truA in trans and, in specific cases, partially suppressed by overexpression of its tRNA substrates, indicating substrate-specific functional dependencies. Analysis of tRNA abundance by northern blotting and AQRNA-seq demonstrates that loss of individual tRNA modification enzymes does not generally lead to widespread tRNA destabilization. Instead, further phenotypic characterization of viable double mutants reveals condition-dependent growth defects influenced by carbon source, temperature and metabolic stress, as well as toxicity associated with overexpression of specific tRNAs. Together, these results reveal a limited but distinct set of genetic interactions among bacterial tRNA modification pathways and highlight the importance of physiological context in uncovering their cellular roles.

microbiology↗

bioRxiv2023

A novel polycistronic method tailored for engineering split GECIs

We assessed the feasibility of using stop-codons as means to obtain polycistronic expression in eukaryotic cells. We show robust bicistronic expression of different open reading frames (ORFs), when these are cloned in-sequence and simply separated by stop codons (in-or out-of-frame), in heterologous expression systems and primary neurons. We further find this method to support polycistronic expression of three stop-codon-separated ORFs in vivo, which guided us to develop a technicolor Genetically-Encoded Functional Rainbow Indicators (GEFRIs) for monitoring cellular morphology and neuronal firing, concomitantly. These findings guided us to develop a new technique we denote SPLIT--Stop-codon mediated Polycistronic Induction in HeTerologous expression systems-- for rapid and easy development of fragmented proteins by the sole use of stop codons. We validated the SPLIT method by generating several new split-GFP variants, then engineer a palette of functional split-GCaMP6s variants and, lastly, generate a split ca2+-probe localized at ER and mitochondria junctions, denoted split-MEGIC. With the use of the probe, we show presence and activity of mito-ER contact sites within individual dendritic spines. Split-MEGIC can thereby be imaged by two-photon excitation in vivo in mice brains and, by standard confocal microscope in transgenic zebrafish larvae. Together, we explore non-canonical translation mechanisms and show these to be highly pervasive in various cell types in vitro and in vivo. We harness translation re-initiation to express multiple ORFs, to engineer rainbow indicators and to swiftly produce functional split-proteins and probes.

neuroscience↗

bioRxiv2022

Two extreme Loss-of-Function GRIN2B-mutations are detrimental to tri-heteromeric NMDAR-function, but rescued by pregnanolone-sulfate

Mutations within various N-methyl-D-aspartate receptor (NMDAR) subunits are tightly associated with severe pediatric neurodevelopmental disorders and encephalopathies (here denoted GRINopathies), for which there are no treatments. NMDARs are tetrameric receptors and can be found at the membrane of neurons in various compositions, namely in di- or tri-heteromeric forms. The GluN2B subunit appears very early in development and, therefore, prenatally this subunit is predominantly found within di-heteromeric receptors, exclusively composed of the GluN1 and GluN2B subunits. Postnatally, however, the GluN2A subunit undergoes rapid increase in expression, giving rise to the appearance of tri-heteromers containing the GluN1, GluN2A and GluN2B-subunits. The latter are emerging as the principal receptor-type postnatally. Despite more than a decade of research of numerous GRINopathies, not much is known regarding the effect of GRIN variants when these are assembled within tri-heteromers. Here, we have systematically examined how two de novo GRIN2B variants (G689C and G689S) affect the function of di- and tri-heteromers. We show that whereas a single mutated subunit readily instigates a dominant negative effect over glutamate affinity of tri-heteromers, it does not dominate other features of the receptor, notably potentiation by pregnanolone-sulfate (PS). This led us to explore PS as a potential treatment for these two severe loss-of-function (LoF) mutations in cultured neurons, in which case we indeed find that the neurosteroid rescues current amplitudes. Together, we present the first report to examine LoF GRIN2B mutations in the context of di- and tri-heteromeric receptors. We also provide the first demonstration of the positive outcome of the use of a GRIN2B-relevant potentiator in the context of tri-heteromers. Our results highlight the importance of examining how different mutations affect features in various receptor subtypes, as these could not have been deduced from observations performed on purely di-heteromers. Together, our study contributes to the ongoing efforts invested towards understanding the pathophysiology of GRINopathies as well as provides insights towards a potential treatment.

neuroscience↗

bioRxiv2020

Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6

Methylation is the most common internal modification in mRNA. While the highly abundant N6-methyladonsine (m6A) modification affects most aspects of mRNA function, the precise functions of the rarer 5-methylcytosine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed that only mRNA methylation sites containing the consensus motif depended on the presence of NSUN6. Furthermore, ribosome profiling demonstrated that NSUN6-specific consensus motifs marked translation termination. However, even though NSUN6-methylated mRNAs were reduced in NSUN6 knockout cells, NSUN6 was dispensable for mouse embryonic development. Thus, our study identifies NSUN6 as methyltransferase targeting mRNA in a sequence- and structure-specific manner.

molecular biology↗

bioRxiv2020

tRNA-modifying enzyme mutations induce codon-specific mistranslation and protein aggregation in yeast

Abstract/SummaryProtein synthesis rate and accuracy are essential for bona fide protein synthesis and proteome homeostasis (proteostasis), however the mRNA translation elongation factors that prevent protein mistranslation, misfolding and aggregation are poorly understood. To address this question, we evaluated the role of 70 yeast tRNA modifying enzyme genes on protein aggregation and used mass spectrometry to identify the aggregated and mistranslated proteins. We show that the mitochondrial tRNA-modifying enzyme Slm3 thiolates the cytoplasmic tRNAs at position 34 and that decreased levels of mcm5s2U34 in SLM3 mutants are compensated by increasing mcm5U34, ncm5U34 and ncm5Um34 levels. In the tRNA gene knockout strains, stress response proteins are overrepresented in protein aggregates and their genes are enriched in codons decoded by tRNAs lacking mcm5U34, mcm5s2U34, ncm5U34, ncm5Um34, modifications. Increased rates of amino acid misincorporation were detected in the yeast ELP1, SLM3 and TRM9 gene knockout mutants at protein sites that specifically mapped to the codons sites that are decoded by the hypomodified tRNAs, demonstrating that U34 tRNA modifications safeguard the proteome from translational errors, misfolding and cellular proteotoxic stress.

molecular biology↗

bioRxiv2020

Cell culture NAIL-MS allows insight into human RNA modification dynamics in vivo

In the last years, studies about the dynamics of RNA modifications are among the most controversially discussed. As the main reason, we have identified the unavailability of a technique which allows to follow the temporal dynamics of RNA transcripts in human cell culture. Here, we present a NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) scheme for efficient stable isotope labeling in both RNA and DNA (>95% within 7 days) in common human cell lines and growth media. Validation experiments reveal that the labeling procedure itself does neither interfere with the isotope dilution MS quantification nor with RNA modification density. We design pulse chase NAIL-MS experiments and apply the new tool to study the temporal placement of modified nucleosides in e.g. tRNAPhe and 18S rRNA. In existing RNAs, we observe a constant loss of modified nucleosides over time which is masked by a post-transcriptional methylation mechanism and thus not detectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing transcripts. Overall, we present a fast and reliable stable isotope labeling strategy which allows a more detailed study of RNA modification dynamics in human cell culture. With cell culture NAIL-MS it is finally possible to study the speed of both modification and demethylation reactions inside human cells. Thus it will be possible to study the impact of external stimuli and stress on human RNA modification kinetics and processing of mature RNA.

biochemistry↗

bioRxiv2020

Production and Purification of Endogenously Modified tRNA-Derived Small RNAs

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.

biochemistry↗

bioRxiv2019

Time-resolved NMR monitoring of tRNA maturation

Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect the introduction of these modifications during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. Here we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNAPhe with time-resolved NMR measurements, we found that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we uncovered a strong hierarchy in the introduction of the T54, {Psi}55 and m1A58 modifications in the T-arm, and demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.

molecular biology↗