Biology subjects

Niehrs, C.

Publications and source records attributed to Niehrs, C..

10 recordsLinked to original sources

bioRxiv2026

Human PARPs modify RNA nucleobases in vitro and in cells

ADP-ribosylation is known as a protein modification, yet recent studies have expanded the range of ADP-ribosyltransferase (ART) substrates to include nucleic acids. tRNA 2'-phosphotransferase 1 (TRPT1) and several PARP family members can modify the 5'-phosphate of single-stranded RNA. Here, we show that PARP10 and PARP15 extend this activity beyond the 5'-phosphate terminus and generate N3-ADP-ribosyl uracil and N1-ADP-ribosyl guanine bases. The base-linked ADP-ribosylation is reversed selectively by the macrodomain-containing hydrolase TARG1. In TARG1 knockout cells, N1-ADP-ribosyl guanine can be detected. Together, these findings establish guanine and uracil ADP-ribose as two novel nucleotide modifications and reveal PARP15 and TARG1 as an enzyme pair which can dynamically regulate guanine ADP-ribosylation in living cells.

biochemistry↗

bioRxiv2025

PARP1 Writes N3-Cytidine ADP-Ribosylation in DNA

Recent evidence indicates that mono - and poly-ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1-deoxyadenosine (N1-dA). Here, we report that PARP1 catalyzes N3-specific ADP-ribosylation of deoxycytidine (N3-dC) in single-stranded DNA. Analogous to N1-dA PARylation, which is prone to spontaneous adenine-to-inosine deamination, N3-dC PARylation promotes cytosine deamination, yielding N3-PARylated-deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC-MS/MS, namely N1-ribosyl-deoxyinosine (N1-r-dI) and N3-ribosyl-deoxyuridine (N3-r-dU). We synthesized both N1-r-dI and N3-r-dU as diagnostic standards and established absolute quantification of base ADP-ribosylations by LC-MS/MS. Quantitative analysis of PARylated dA and dC in ssDNA reveals pronounced sequence preferences of PARP1. Removal of these base modifications differs markedly, since ADP-ribose glycohydrolase TARG1 removes PAR from both dA and dC, whereas PARG acts exclusively on dA. Our results establish cytidine ADP-ribosylation as a novel DNA modification, with potential roles in DNA metabolism, epigenetic regulation, or genome stability.

biochemistry↗

bioRxiv2025

Secretion and signaling properties of Wnt11 and Wnt3 are determined by their N-terminus

Wnt proteins are evolutionarily conserved signaling molecules that control cell-cell communication in health and disease. While different Wnt proteins share a requirement for palmitoleoylation and the cargo receptor Evi/Wls for their secretion, they interact with diverse signaling receptors and co-receptors to elicit distinct outcomes in signal-receiving cells. The molecular mechanisms underlying this contrast between conserved post-translational modification and secretion and diverse receptor interactions remain poorly understood. Here, we demonstrate that Wnt11 maintains partial secretion capability in the absence of Evi/Wls. Unexpectedly, the secreted protein is also non-palmitoleoylated, yet retains its ability to activate downstream signaling in receiving cells through the Frizzled 6 receptor. We identify the N-terminus as the mediator of this unusual behavior, as Wnt3, which strictly requires Evi/Wls, becomes Evi/Wls-independent when its N-terminus is replaced with that of Wnt11. In different cell lines and in Xenopus laevis embryos, the chimeric Wnt11Nterm-Wnt3 protein recapitulates known Wnt11 phenotypes, demonstrating that the N-terminal portion of Wnt11 is sufficient to determine signaling specificity. Our findings reveal novel determinants of Wnt protein specificity and provide mechanistic insights into the molecular basis of Wnt secretion and downstream signaling.

cell biology↗

bioRxiv2022

ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation

Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention but options to target cell surface proteins and receptors remain limited. Here we introduce ROTACs, bispecific WNT- and BMP signaling-disabled R-spondin (RSPO) chimeras, which leverage the specificity of these stem cell growth factors for ZNRF3/RNF43 E3 transmembrane ligases, to target degradation of transmembrane proteins. As proof of concept, we targeted the immune checkpoint protein programmed death ligand 1 (PD-L1), a prominent cancer therapeutic target, with a bispecific RSPO2 chimera, R2PD1. The R2PD1 chimeric protein bound PD-L1 and at picomolar concentration induced its lysosomal degradation. In three melanoma cell lines, R2PD1 induced between 50-90% PD-L1 protein degradation. PD-L1 degradation was strictly dependent on ZNRF3/RNF43. We conclude that signaling-disabled ROTACs represent a novel strategy to target cell surface proteins for degradation.

bioengineering↗

bioRxiv2022

Primary Cilia are WNT Transducing Organelles whose Biogenesis is Regulated by a WNTPP1 axis

WNT signalling is of paramount importance in development, stem cell maintenance, and disease. WNT ligands typically signal via receptor activation at the plasma membrane to induce {beta}-catenin-dependent gene activation. Here we show that in primary cilia, WNT receptors relay a WNT/GSK3 signal that {beta}-catenin-independently promotes ciliogenesis. Innovations supporting this conclusion are monitoring acute WNT co-receptor activation (phospho-LRP6) and identifying and mutating the LRP6 ciliary targeting sequence. Ciliary WNT signalling inhibits protein phosphatase 1 (PP1) activity, a negative regulator of ciliogenesis, by decommissioning GSK3-mediated phosphorylation of the PP1 regulatory inhibitor subunit PPP1R2. Accordingly, deficiency of WNT/GSK3 signalling by depletion of cyclin Y and cyclin-Y-like protein 1 induces widespread primary cilia defects in mouse embryonic neuronal precursors, kidney proximal tubules, and adult mice preadipocytes. We conclude that primary cilia are WNT PP1 signalling organelles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/519813v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@4e8f4dorg.highwire.dtl.DTLVardef@1a246f6org.highwire.dtl.DTLVardef@e0df10org.highwire.dtl.DTLVardef@154a139_HPS_FORMAT_FIGEXP M_FIG A Localized WNT PP1 Signalling Axis Promotes Ciliogenesis The WNT co-receptor LRP6 localizes to the ciliary membrane, where it is phospho-primed via a CCNY/L1-dependent CDK (not shown). WNT signalling inhibits GSK3 (not shown) and leads to inhibition of Protein phosphatase 1, a negative regulator of ciliogenesis. Right, CCNY/L1 deficiency disrupts the WNT PP1 signalling axis, leading to ciliary defects. C_FIG

cell biology↗

bioRxiv2022

Mammalian N1-adenosine PARylation is a reversible DNA modification

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. We conclude that N1-adenosine PARylation is a novel, reversible DNA modification.

molecular biology↗

bioRxiv2022

DNA methylation clocks for clawed frogs reveal evolutionary conservation of epigenetic ageing

DNA methylation-based biomarkers of ageing (epigenetic clocks) have been developed for many mammals, but not yet for amphibian species. We generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis), from adult tissues, whole embryos, and tadpoles. We used an array platform designed for CpGs that are highly conserved in mammals to build multiple DNA methylation-based estimators of age for Xenopus. We found that dual species clock could be developed that apply to both humans and frogs (human-clawed frog clocks), and whose high accuracy supports that epigenetic ageing processes are evolutionary conserved outside mammals. Crossing vast evolutionary distances, we characterize age-related CpGs in Xenopus and mammalian species. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. As in human clocks, positively age-related CpGs are associated with Polycomb repressive complex 2 (PRC2) target sites. Negative age-related CpGs are associated with genes involved in synaptic transmission. We conclude that signatures of epigenetic ageing are evolutionary conserved between frogs and mammals and that the associated genes relate to neural-developmental processes, altogether opening opportunities to employ Xenopus as a model organism to study development and ageing.

developmental biology↗

bioRxiv2021

DEAD-box RNA Helicases Act as Nucleotide Exchange Factors for Casein Kinase 2

DDX RNA helicases promote RNA processing but DDX3X is also known to activate casein kinase 1 {varepsilon} (CK1{varepsilon}). Here we show that not only is protein kinase stimulation a latent property of other DDX proteins towards CK1{varepsilon}, but that this extends to casein kinase 2 (CK22) as well. CK22 enzymatic activity is stimulated by a variety of DDX proteins and we identify DDX1/24/41/54 as physiological activators required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X reveals that CK1 and CK2 kinase stimulation engages its RNA binding-but not catalytic motifs. Mathematical modelling of enzyme kinetics and stopped-flow spectroscopy converge that DDX proteins function as nucleotide exchange factor towards CK22 that reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as a new principle in kinase regulation and an evolved function of DDX proteins.

biochemistry↗

bioRxiv2020

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimised miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses are calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification. HighlightsO_LImiCLIP2 produces complex libraries to map m6A RNA modifications C_LIO_LIMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sites C_LIO_LIMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KO C_LIO_LIm6A modifications occur outside of DRACH motifs and associate with alternative splicing C_LI

genomics↗

bioRxiv2020

R-spondins are BMP receptor antagonists in early embryonic development

BMP signalling plays key roles in development, stem cells, adult tissue homeostasis, and disease. How BMP receptors are extracellularly modulated and in which physiological context, is therefore of prime importance. R-spondins (RSPOs) are a small family of secreted proteins that co-activate WNT signalling and function as potent stem cell effectors and oncogenes. Evidence is mounting that RSPOs act WNT-independently but how and in which physiological processes remains enigmatic. Here we show that RSPO2 and RSPO3 also act as BMP antagonists. RSPO2 is a high affinity ligand for the type I BMP receptor BMPR1A/ALK3, and it engages ZNRF3 to trigger internalization and degradation of BMPR1A. In early Xenopus embryos, Rspo2 is a negative feedback inhibitor in the BMP4 synexpression group and regulates dorsoventral axis formation. We conclude that R-Spondins are bifunctional ligands, which activate WNT- and inhibit BMP signalling via ZNRF3, with implications for development and cancer.

developmental biology↗