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Detection of nucleic acids

Mechanisms and functional consequences of Y-DNA immune sensing (A01) 

TR_A01.4

Project leaders: Prof. Gunther Hartmann and Dr. Florian Schmidt 

Innate immune sensing of DNA is a mainstay of antiviral defense. Dysregulation can not only impair antiviral immunity but can lead to erroneous detection of endogenous DNA molecules, resulting in autoimmune diseases. Therefore it is of utmost importance to understand the molecular mechanisms that govern DNA detection and that coordinate the downstream signaling events. Cytosolic DNA is sensed in many cell types by the cGAS/STING axis to induce type I interferon (IFN). Additionally, sentinal cells like Macrophages and dendritic cells assemble DNA-dependent inflammasomes. We are interested in the functional interaction of the type I IFN and the inflammasome pathways on the level of receptor, ligand, and signaling. To elucidate the molecular mechanism and functional implications of DNA immune sensing by cGAS, AIM2, and other cytosolic DNA receptors, we will use well-defined synthetic DNA ligands. We will use our unique expertise do generate alpaca nanobodies as innovative tools to study signaling pathways in their endogenous settings. Reporter constructs will allow us to quantify the activation of type I IFN and inflammasome pathways and their crosstalk in single-cell resolution. We will further identify novel cellular adaptors required, and delineate which additional short DNA species activate the respective sensors in the context of DNA damage. The new insights we acquire will help to clarify the immunopathogenesis of autoinflammatory conditions and aide the development of novel treatment strategies.


Role of RNA modifications in infection and immune tolerance (A02)  

TR_A02.4

Project leader: Prof. Stefan Bauer 

The innate immune system relies on Toll-like receptors and RIG-I like helicases to sense bacterial and viral RNA. RNA modifications such as 2’-O-ribose / base methylation or the occurrence of pseudouridine negatively modulate the immune sensing of RNA. We will investigate the role of bacterial tRNA methylase trmH in immune evasion and characterize the importance of the eukaryotic RNA modifying enzymes fibrillarin, TAR binding protein 1, and dyskerin pseudouridine synthetase 1 (DKC1) for the generation of modified self RNA preventing immune activation and induction of autoimmunity.


Recognition of pathogenic RNA in mosquitoes (A04)

TR_A04.4

Project leaders: PD Dr. Beate Kümmerer and Prof. Martin Schlee 

RNA virus based mosquito-borne diseases cause substantial morbidity worldwide. Innate antiviral RNA receptors are well defined in vertebrates but hardly studied in insects. We found that flavivirus replication is impaired in vertebrate and mosquito cells if mRNA cap N1-2'O-methylation is lacking, indicating conserved virus RNA recognition motifs. Screening RNA recognition motifs as stimuli or bait in interactome approaches, we aim to identify RNA receptors and characterize antiviral responses in mosquitoes. In a virus mutagenesis reverse genetics approach, we aim to identify viral antagonists of mosquito innate RNA receptor pathways and their interacting antiviral host proteins.


Structural mechanisms of self-nucleic acid sensing by cGAS (A05)

TR_A05.4

Project leader: Prof. Karl-Peter Hopfner  

We will interrogate how cGAS detects DNA of self-origin, in particular, damaged DNA, cytosolic chromatin, and mitochondrial DNA. We recently showed that cGAS forms higher-order ladder-like structures on long and U-turn DNA, suggesting that stabilization of cGAS dimers and multimers is critical for DNA sensing. Using structural, biochemical and cell-based studies, we aim at revealing now whether and how branched nucleic acids, cGAS’ N-terminal domain, and HMGB/HIN200 host factors help the formation of active cGAS dimer and multimers and therefore understand mechanisms of self-sensing.


 Recognition and consequences of RNA-G4 structures during viral infection (A23)

TR_A23.5

Project leader: Prof. Dr. Katrin Paeschke

G-quadruplexes (G4s) are non-canonical nucleic acid structures that form within a specific guanine-rich motif. G4-structures are found in all genomes investigated to date. In the context of viral infection, they were linked to the viral replicative lifespan and it is assumed that viruses G4 regulation strongly depends on host proteins. Further, accumulating RNA-G4 structures activate cellular stress signals and an innate immune response. With transcriptome-wide experiments (PAR-CLIP, RNA-seq, Ribo-seq) and molecular approaches using reporter assays, microscopy and qPCR analyses we want to investigate the role of RNA-G4 structures in both self and viral RNA in the immune response. We are interested in how these structures are recognized and what consequences arise for the host cell. Therefore we will characterize the nature and mechanism of the G4-induced immune response by using different G4 stimuli. We will shed light on how RNA-G4 structures challenge human cells and how these nucleic acid structures are sensed and regulated and describe the effects of the accumulation of G4-structures has on host cells. We will perform in vivo and in vitro assays to identify novel RNA-G4 binders. Altogether, this project aims to characterize the role of RNA-G4 structures as a novel regulator of the immune response to self and viral RNA.


 

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