Biology subjects

Carell, T.

Publications and source records attributed to Carell, T..

18 recordsLinked to original sources

bioRxiv2026

The Escherichia coli Radical SAM Enzyme YhcC Substitutes for the FAD-Dependent Oxidase Activity of MnmC in 5-Methylaminomethyl-2-Thiouridine tRNA Modification Under Anaerobic Conditions

tRNA wobble uridines are heavily modified to influence anticodon-codon pairing and tune anticodon stem-loop structure for efficient, accurate translation. Many bacteria and some archaea modify wobble uridines with either a 5-carboxymethylaminomethyl (cmnm5) or 5-methylaminomethyl (mnm5) group, often together with a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further converted to mnm5U by non-orthologous enzymes in different lineages. Escherichia coli uses the bifunctional enzyme MnmC to demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U whereas Bacillus subtilis relies on the radical SAM (rSAM) enzyme MnmL and the stand-alone methylase MnmM. Although E. coli and related bacteria encode homologs of MnmL, the function of the E. coli homolog, YhcC, remained unknown. Here, we show that YhcC is required for cmnm5s2 U demodification in vivo during anaerobic growth, whereas the equivalent MnmC-dependent reaction requires O2 and occurs only aerobically. In vitro, purified [4Fe-4S]-reconstituted YhcC binds tRNA and catalyzes nm5s2U-tRNA synthesis from cmnm5s2U-tRNA. Together, these results define the previously unknown function of the E. coli rSAM enzyme YhcC and demonstrate that it replaces MnmC under anaerobic conditions to generate nm5s2U. These parallel pathways reveal how E. coli maintains synthesis of a critical wobble-base modification under both aerobic and anaerobic growth conditions.

biochemistry↗

bioRxiv2025

Increasing TET expression and 5-hydroxymethylcytosine formation by a carbocyclic 5-aza-2'-deoxy-cytidine antimetabolite

Ten-eleven translocation (TET) enzymes are critical epigenetic regulators, which oxidize the methylated cytosine nucleobase 5-methyl-dC (5mdC) in the genome to 5-hydroxymethyl-dC (5hmdC) in an -ketoglutarate-dependent manner. Because the presence of 5mdC in the promoter region of a given gene silences its expression, this oxidation goes in hand with the reactivation of such silenced genes. In different highly aggressive cancers such as acute myeloid leukemia (AML) and glioblastoma, loss of TET enzyme function and therefore reduced 5hmdC levels pave the way for tumor development. Impairment of TET activity can occur through metabolic inhibition, through loss-of-function mutations in TET genes themselves, and finally through suppression of TET-expression via epigenetic silencing. Reactivation of TET enzyme expression represents a major aim of epigenetic cancer therapy. Here we show that the carbocyclic antimetabolite 5-aza-2deoxycytidine (cAzadC), which is supposed to suppress the methylation of DNA during replication, leads to a substantial increase of TET2 expression and strongly increasing 5hmdC levels. We show that the treatment with cAzadC goes in hand with the broad reactivation of the cellular anti-tumor responses. With patient-derived xenograft AML-mouse models, we show that this translates into a strongly improved anti-cancer effect in vivo.

cancer biology↗

bioRxiv2025

ALKBH1 activity in vitro and human cell lines by isotope dilution mass spectrometry

AlkB homolog 1 (ALKBH1) is a member of the AlkB family of Fe(II) and -ketoglutarate (-KG)-dependent dioxygenases, known for its enzymatic activity on various nucleic acid substrates. Its known targets include N1-methyladenosine (m1A), N6-methyladenosine (m6A), N3-methylcytidine (m3C), and 5-methylcytosine (m5C) in RNA, as well as N6-methyladenine (N6-mA, 6mA) in DNA and the histone protein H2A. Moreover, dysregulation or dysfunction of ALKBH1 has been implicated in a broad spectrum of human diseases. In order to shed further light on the substrate scope and role of ALKBH1, we used quantitative mass spectrometry to assess its activity in vitro and in human cell lines. To study ALKBH1 activity on defined substrates in vitro, we enzymatically generated tRNAs specifically carrying the m3C32 (tRNA-ThrUGU and tRNA-SerUCN) and i6A37 (tRNA-SerUCN) modifications. Here we show that ALKBH1 reduces m3C, m1A and m5C in vitro in total extracts of tRNAs, but does not impact on rRNA modifications in human cell lines. However, upon overexpression or siRNA-mediated knock-down of alkbh1 in human cell lines no impact on the modification of total tRNA extracts or specifically enriched RNAs could be observed. In addition, varying the glucose and fetal bovine serum (FBS) concentration in the growth medium of HEK293T cells, in combination with alkbh1 siRNA-mediated knock-down, also shows no impact on the tRNA modification spectra. Based on our data, we conclude that in human cells lines grown under optimal conditions ALKBH1 does not play an important role in the demethylation of tRNAs.

biochemistry↗

bioRxiv2025

Crosstalk between the Methyl-Cytosine Dioxygenase TET3 and the Methyl-CpG-binding protein MECP2 Controls Neuronal Maturation

Active DNA demethylation depends on Ten-Eleven-Translocation (TET) enzymes, which oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC) and further derivatives. Mutations in TET3, encoding the predominant neuronal isoform, lead to Beck-Fahrner syndrome, a neurodevelopmental disorder. Using human iPSC-derived neurons, we show that TET3 is dispensable for neuronal specification but critical for subsequent maturation. Differentiating TET3-deficient neurons exhibit delayed transcriptional and proteomic transitions, altered synaptic signatures, and impaired network activity, indicating delayed functional maturation. Mechanistically, we identified an interaction between TET3 and the mC/hmC-binding protein MECP2, pathogenic variants of which cause Rett syndrome. MECP2 negatively regulates TET3 activity, as demonstrated in functional assays and by inverse hmC patterns in MECP2- and TET3-deficient neurons. Despite this, MECP2- and TET3-deficient neurons exhibit highly similar phenotypes later in differentiation. Our findings uncover a functional interplay between TET3 and MECP2 that coordinates DNA methylation and chromatin dynamics during neuronal maturation, suggesting a shared pathogenic mechanism in Beck-Fahrner and Rett syndromes.

neuroscience↗

bioRxiv2025

Towards One-Step and Universal Synthesis of Hypermodified Uracil Nucleosides: acp3U and cmnm5U

Hypermodified nucleosides have been recently in the center of scientific attention. They represent a unique group of nucleosides with an alternated structure, such as the addition of functional groups or amino acids. Their distinctive structures and positions in RNAs are crucial for the processes of translation and stability. High demand of oligonucleotides bearing those hypermodified nucleobases lead us to the development of a single step synthesis of acp3U phosphoramidite, as acp3U has been recognized as an important molecule for the structural integrity of tRNA and native immunity. We also present a novel synthesis of cmnm5U phosphoramidite and its incorporation into an oligonucleotide from a universal starting material, allowing a transformation into at least two other hypermodified nucleosides.

molecular biology↗

bioRxiv2025

Biochemical engineering of 5hmdC-DNA using a Tet3 double-mutant

5-Hydroxymethyl-2-deoxycytidine (5hmdC) is an important epigenetic marker involved in gene regulation and DNA demethylation. It has potential use as a biomarker for cancer and other diseases due to its significant depletion in various cancers and disease models. This research aimed to develop a reliable and efficient method for generating 5hmdC-containing DNA, addressing limitations in existing techniques. We created a Tet3 stalling mutant that converts 5-methyl-2'-deoxycytidine (5mdC) into a mixture of 5hmdC and 5-formyl-2'-deoxycytidine (5fdC), followed by a reduction step to convert 5fdC to 5hmdC, ensuring a pure 5hmdC state within the CpG context. This method can convert any PCR product, synthetic oligos, and entire genomes into 5hmdC-modified DNA. The principal results demonstrate high specificity and efficiency, providing a robust tool for epigenetic research, cancer diagnostics, and protein binding assays. Additionally, our technique offers 5hmdC-DNA for functional studies and as standards for diagnostic assays.

biochemistry↗

bioRxiv2024

Membrane bound geranylated RNAs establish a primitive peptide synthesis system

The origin of an RNA-based translation system necessitates a specific interaction of certain RNAs with defined amino acids. This must have happened in a protocellular environment where these molecules were concentrated so that a connection between the encoding RNA and the amino acids could be established that allowed the formation of peptides. A model of how such a system could have evolved does not exist. Here we show that geranylated non-canonical nucleotides that are potential fossils in an early RNA world, allow RNA to anchor to lipid membranes. This creates RNA-geranylating lipids on which a primitive peptide synthesis can then operate with rudimentary chemoselectivity. The system creates a protocellular model of how RNAs and amino acids could have been mutually selected based on their physicochemical properties.

molecular biology↗

bioRxiv2024

Depletion of m6A-RNA in Escherichia coli reduces the infectious potential of T5 bacteriophage

N6-methyladenosine (m6A) is the most abundant internal modification of mRNA in eukaryotes that plays, among other mechanisms, an essential role in virus replication. However, the understanding of m6A RNA modification in prokaryotes, especially in relation to phage replication, is limited. To address this knowledge gap, we investigated the effects of m6A RNA modification on phage replication in two model organisms: Vibrio campbellii BAA-1116 (previously V. harveyi BB120) and Escherichia coli MG1655. An m6A-RNA depleted V. campbellii mutant ({Delta}rlmF{Delta}rlmJ) did not differ from the wild type in the induction of lysogenic phages or in susceptibility to the lytic Virtus phage. In contrast, the infection potential of the T5 phage, but not that of other T phages or the lambda phage, was reduced in an m6A-RNA depleted E. coli mutant ({Delta}rlmF{Delta}rlmJ) compared to the wild type. This was shown by a lower efficiency of plaquing and a higher percentage of surviving cells. There were no differences in T5 phage adsorption rate, but the mutant exhibited a 5 min delay in the rise period during the one-step growth curve. This is the first report demonstrating that E. coli cells with lower m6A RNA levels have a higher chance of surviving T5 phage infection. ImportanceThe importance of RNA modifications has been thoroughly studied in the context of eukaryotic viral infections. However, their role in bacterial hosts during phage infections is largely unexplored. Our research delves into this gap by investigating the effect of host m6A-RNA modifications during phage infection. We found that an E. coli mutant depleted of m6A-RNA is more resistant to T5 infection than the wild type. This finding emphasizes the need to further investigate how RNA modifications affect the fine-tuned regulation of individual bacterial survival in the presence of phages to ensure population survival.

microbiology↗

bioRxiv2024

Generation of densely labeled oligonucleotides for the detection of small genomic elements

The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA-probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.

cell biology↗

bioRxiv2022

Human TRMT2A methylates different components of the translation machinery and contributes to translation fidelity

Methyl-5-uridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme of m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by TRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with crosslinking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that TRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of TRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

biochemistry↗

bioRxiv2022

Epigenetic anti-cancer treatment with a stabilized carbocyclic Decitabine analogue

5-Aza-2-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

cancer biology↗

bioRxiv2021

1H NMR chemical exchange techniques reveal local and global effects of oxidized cytosine derivatives

5-carboxycytosine (5caC) is a rare epigenetic modification found in nucleic acids of all domains of life. Despite its sparse genomic abundance, 5caC is presumed to play essential regulatory roles in transcription, maintenance and baseexcision processes in DNA. In this work, we utilize nuclear magnetic resonance (NMR) spectroscopy to address the effects of 5caC incorporation into canonical DNA strands at multiple pH and temperature conditions. Our results demonstrate that 5caC has a pH-dependent global destabilizing and a base-pair mobility enhancing local impact on dsDNA, albeit without any detectable influence on the ground-state B-DNA structure. Measurement of hybridization thermodynamics and kinetics of 5caC-bearing DNA duplexes highlighted how acidic environment (pH 5.8 and 4.7) destabilizes the double-stranded structure by ~10-20 kJ mol-1 at 37 {degrees}C when compared to the same sample at neutral pH. Protonation of 5caC results in a lower activation energy for the dissociation process and a higher barrier for annealing. Studies on conformational exchange on the s time scale regime revealed a sharply localized base-pair motion involving exclusively the modified site and its immediate surroundings. By direct comparison with canonical and 5-formylcytosine (5fC)-edited strands, we were able to address the impact of the two most oxidized naturally occurring cytosine derivatives in the genome. These insights on 5caCs subtle sensitivity to acidic pH contribute to the long standing questions of its capacity as a substrate in base excision repair processes and its purpose as an independent, stable epigenetic mark.

biophysics↗

bioRxiv2021

Synthesis and Structure Elucidation of the Human tRNA Nucleoside Mannosyl-Queuosine

Queuosine (Q) is a structurally complex, non-canonical RNA nucleoside. It is present in many eukaryotic and bacterial species, where it is part of the anticodon loop of certain tRNAs. In higher vertebrates, including humans, two further modified queuosine-derivatives exist - galactosyl-(galQ) and mannosyl-queuosine (manQ). The function of these low abundant hypermodified RNA nucleosides remains unknown. While the structure of galQ was elucidated and confirmed by total synthesis, the reported structure of manQ still awaits confirmation. By combining total synthesis and LC-MS-co-injection, together with a metabolic feeding study of labelled hexoses, we show here that the natural compound manQ isolated from mouse liver deviates from the literature-reported structure. The chemical structure of the natural product manQ features a novel -allyl connectivity. The data reported here shows that the yet unidentified glycosylases that attach galactose and mannose to the Q-base have different constitutional connectivity preferences. Knowing the correct structure of manQ will now pave the way towards further elucidation of its biological function.

biochemistry↗

bioRxiv2020

TENT4A poly(A) polymerase regulates translesion DNA synthesis and is mutated in endometrial cancer

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here we focus on its multilayer regulation of translesion DNA synthesis (TLS), in which DNA lesions are bypassed by error-prone DNA polymerases. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase {eta} and RAD18 E3 ligase, which guides the polymerase to replication stalling sites, and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD, and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase {eta}, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

molecular biology↗

bioRxiv2019

Oxidative and non-oxidative active turnover of genomic methylcytosine in distinct pluripotent states

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known and the underlying pathways are poorly understood. Here we use metabolic labelling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic methylcytidine (mdC), hydroxymethylcytidine (hmdC) and formylcytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on Tet-mediated mdC oxidation, we unveiled an additional mdC oxidation-independent turnover process based on DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage committed cells. Thus, in pluripotent cells active mdC turnover involves both mdC oxidation-dependent and -independent processes.

cell biology↗

bioRxiv2019

DNA hydroxymethylation reveals transcription regulation networks and prognostic signatures in multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy that remains challenging to cure despite a substantially improving median survival. During the last decade, DNA copy number variation and gene expression studies have described the pathology and its heterogeneity among patients. Epigenetic modifications play important roles in MM, but they are rarely associated with clinical aspects of the disease. In this epigenomics study, we produced quantifications of genomic 5-methylcytosine (5mC) and of 5-hydroxymethylcytosine (5hmC) as well as genome-wide maps of hydroxymethylation to analyse myeloma cells taken from a cohort of 40 newly diagnosed and homogeneously treated patients. We found 5hmC to be globally depleted in MM compared to normal plasma cells, as well as being reduced in advanced clinical stages of the disease. From the hydroxymethylome data, we observed that remaining 5hmC is organised in large peak clusters and is associated with well-known disease-related genes. Based on their signal correlation, these 5hmC peak clusters can be gathered in 2 regulation networks involving core transcription factors such as IRF4, MYC, PRDM1 and TCF3. By performing paired hydroxymethylomes at diagnosis and at relapse, we found the disease progression to be heterogeneous and patient-specific. We found that the location of 5hmC at tumor suppressor TP53INP1 is associated with better outcome while global high level of 5hmC tends to be associated with better overall survival. Together, our study suggests that 5hmC provides new biological insights of the disease severity and progression, and can be used for retrospective studies.\n\nKey PointsO_LI5hmC is globally depleted in MM and even more in advanced stages of the disease.\nC_LIO_LI5hmC is locally detected at transcriptionally active regions of MM where its presence can be associated with survival.\nC_LI

cancer biology↗

bioRxiv2018

TET1 drives global DNA demethylation via DPPA3-mediated inhibition of maintenance methylation

Genome-wide DNA demethylation is a unique feature of mammalian development and naive pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naive pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.

developmental biology↗

bioRxiv2018

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

The TET-oxidized cytosine derivatives, 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), are considered DNA demethylation intermediates as well as stable epigenetic marks in mammals. We compared modified cytosine and enzyme levels in TET-knockout cells during naive pluripotency exit and found distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation. The divergent modified cytosine levels argue for independent consecutive oxidation steps in vivo with broad implications for epigenetic regulation.

cell biology↗