TLR8 Is a Sensor of RNase T2 Degradation Products.

TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment, yet its mode of action is poorly understood. TLR8 engages two distinct ligand binding sites to sense RNA degradation products, although it remains unclear how these ligands are formed in cellulo in the context of complex RNA molecule sensing. Here, we identified the lysosomal endoribonuclease RNase T2 as a non-redundant upstream component of TLR8-dependent RNA recognition. RNase T2 activity is required for rendering complex single-stranded, exogenous RNA molecules detectable for TLR8. This is due to RNase T2's preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Thus-generated molecules constitute agonistic ligands for the first and second binding pocket of TLR8. Together, these results establish the identity and origin of the RNA-derived molecular pattern sensed by TLR8.

Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

Human TRMT2A methylates tRNA and contributes to translation fidelity.

5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for 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 hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

tRNA modification profiles in obligate and moderate thermophilic bacilli.

Transfer RNAs (tRNAs) are the most ancient RNA molecules in the cell, modification pattern of which is linked to phylogeny. The aim of this study was to determine the tRNA modification profiles of obligate (Anoxybacillus, Geobacillus, Paragebacillus) and moderate (Bacillus, Brevibacillus, Ureibacillus, Paenibacillus) thermophilic aerobic bacilli strains to find out its linkage to phylogenetic variations between species. LC-MS was applied for the quantification of modified nucleosides using both natural and isotopically labeled standards. The presence of m2A and m7G modifications at high levels was determined in all species. Relatively high level of i6A and m5C modification was observed for Paenibacillus and Ureibacillus, respectively. The lowest level of Cm modification was found in Bacillus. The modification ms2i6A and m1G were absent in Brevibacillus and Ureibacillus, respectively, while modifications Am and m22G were observed only for Ureibacillus. While both obligate and moderate thermophilic species contain Gm, m1G and ms2i6A modifications, large quantities of them (especially Gm and ms2i6A modification) were detected in obligate thermophilic ones (Geobacillus, Paragebacillus and Anoxybacillus). The collective set of modified tRNA bases is genus-specific and linked to the phylogeny of bacilli. In addition, the dataset could be applied to distinguish obligate thermophilic bacilli from moderate ones.

Novel Poxin Stable cGAMP-Derivatives Are Remarkable STING Agonists.

2',3'-cGAMP is a cyclic A- and G-containing dinucleotide second messenger, which is formed upon cellular recognition of foreign cytosolic DNA as part of the innate immune response. The molecule binds to the adaptor protein STING, which induces an immune response characterized by the production of type I interferons and cytokines. The development of STING-binding molecules with both agonistic as well as antagonistic properties is currently of tremendous interest to induce or enhance antitumor or antiviral immunity on the one hand, or to treat autoimmune diseases on the other hand. To escape the host innate immune recognition, some viruses encode poxin endonucleases that cleave 2',3'-cGAMP. Here we report that dideoxy-2',3'-cGAMP (1) and analogs thereof, which lack the secondary ribose-OH groups, form a group of poxin-stable STING agonists. Despite their reduced affinity to STING, particularly the compound constructed from two A nucleosides, dideoxy-2',3'-cAAMP (2), features an unusually high antitumor response in mice.

Synthesis of Galactosyl-Queuosine and Distribution of Hypermodified Q-Nucleosides in Mouse Tissues.

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNAHis , tRNAAsn , tRNATyr , and tRNAAsp . It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNATyr and tRNAAsp of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl-queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q-family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q-nucleoside family.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.

5-Formyl-2'-deoxycytosine (fdC) is a recently discovered epigenetic base in the genome of stem cells, with yet unknown functions. Sequencing data show that the base is enriched in CpG islands of promoters and hence likely involved in the regulation of transcription during cellular differentiation. fdC is known to be recognized and excised by the enzyme thymine-DNA-glycosylase (Tdg). As such, fdC is believed to function as an intermediate during active demethylation. In order to understand the function of the new epigenetic base fdC, it is important to analyze its formation and removal at defined genomic sites. Here, we report a new method that combines sequence-specific chemical derivatization of fdC with droplet digital PCR that enables such analysis. We show initial data, indicating that the repair protein Tdg removes only 50 % of the fdCs at a given genomic site, arguing that fdC is a semipermanent base.

Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues.

The absolute levels of 5-hydroxymethylcytosine (hmC) and 5-methylcytosine (mC) in human brain tissues at various ages were determined. Additionally, absolute levels of 5-formylcytosine (fC) in adult individuals and cytosine modification levels in sorted neurons were quantified. These data were compared with age-related fC, hmC, and mC levels in mouse brain samples. For hmC, an initial steady increase is observed, which levels off with age to a final steady-state value of 1.2 % in human brain tissue. This level is nearly twice as high as in mouse cerebral cortex. In contrast, fC declines rapidly with age during early developmental stages, thus suggesting that while hmC is a stable epigenetic mark, fC is more likely an intermediate of active DNA demethylation during early brain development. The trends in global cytosine modification dynamics during the lifespan of an organism are conserved between humans and mice and show similar patterns in different organs.

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA.

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

Depletion of pyruvate kinase (PK) activity causes glycolytic intermediate imbalances and reveals a PK-TXNIP regulatory axis.

OBJECTIVE: Cancer cells convert more glucose into lactate than healthy cells, what contributes to their growth advantage. Pyruvate kinase (PK) is a key rate limiting enzyme in this process, what makes it a promising potential therapeutic target. However, currently it is still unclear what consequences the inhibition of PK has on cellular processes. Here, we systematically investigate the consequences of PK depletion for gene expression, histone modifications and metabolism. METHODS: Epigenetic, transcriptional and metabolic targets were analysed in different cellular and animal models with stable knockdown or knockout of PK. RESULTS: Depleting PK activity reduces the glycolytic flux and causes accumulation of glucose-6-phosphate (G6P). Such metabolic perturbation results in stimulation of the activity of a heterodimeric pair of transcription factors MondoA and MLX but not in a major reprogramming of the global H3K9ac and H3K4me3 histone modification landscape. The MondoA:MLX heterodimer upregulates expression of thioredoxin-interacting protein (TXNIP) - a tumour suppressor with multifaceted anticancer activity. This effect of TXNIP upregulation extends beyond immortalised cancer cell lines and is applicable to multiple cellular and animal models. CONCLUSIONS: Our work shows that actions of often pro-tumorigenic PK and anti-tumorigenic TXNIP are tightly linked via a glycolytic intermediate. We suggest that PK depletion stimulates the activity of MondoA:MLX transcription factor heterodimers and subsequently, increases cellular TXNIP levels. TXNIP-mediated inhibition of thioredoxin (TXN) can reduce the ability of cells to scavenge reactive oxygen species (ROS) leading to the oxidative damage of cellular structures including DNA. These findings highlight an important regulatory axis affecting tumour suppression mechanisms and provide an attractive opportunity for combination cancer therapies targeting glycolytic activity and ROS-generating pathways.

Low values of 5-hydroxymethylcytosine (5hmC), the "sixth base," are associated with anaplasia in human brain tumors.

5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.

Structure and function of noncanonical nucleobases.

DNA and RNA contain, next to the four canonical nucleobases, a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m(5) dC, hm(5) dC, f(5) dC und ca(5) dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified non-canonical nucleosides in both DNA and RNA.

Isotope-based analysis of modified tRNA nucleosides correlates modification density with translational efficiency.

Useful diversity: Quantification of modified tRNA nucleobases in different murine and porcine tissues reveals a tissue-specific overall modification content. The modification content correlates with rates of protein synthesis in vitro, suggesting a direct link between tRNA modification levels and tissue-specific translational efficiency.

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

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 experiments, 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. Our data show that manQ features an α-allyl connectivity of its sugar moiety. The yet unidentified glycosylases that attach galactose and mannose to the Q-base therefore have a maximally different constitutional connectivity preference. Knowing the correct structure of manQ will now pave the way towards further elucidation of its biological function.

Intercellular cGAMP transmission induces innate immune activation and tissue inflammation in Trex1 deficiency.

Intercellular transmission of the second messenger 2',3'-cGAMP, synthesized by the viral DNA sensor cGAMP synthase (cGAS), is a potent mode of bystander activation during host defense. However, whether this mechanism also contributes to cGAS-dependent autoimmunity remains unknown. Here, using a murine bone marrow transplantation strategy, we demonstrate that, in Trex1 -/- -associated autoimmunity, cGAMP shuttling from radioresistant to immune cells induces NF-κB activation, interferon regulatory factor 3 (IRF3) phosphorylation, and subsequent interferon signaling. cGAMP travel prevented myeloid cell and lymphocyte death, promoting their accumulation in secondary lymphoid tissue. Nonetheless, it did not stimulate B cell differentiation into autoantibody-producing plasmablasts or aberrant T cell priming. Although cGAMP-mediated bystander activation did not induce spontaneous organ disease, it did trigger interface dermatitis after UV light exposure, similar to cutaneous lupus erythematosus. These findings reveal that, in Trex1-deficiency, intercellular cGAMP transfer propagates cGAS signaling and, under conducive conditions, causes tissue inflammation.

DNA hydroxymethylation is associated with disease severity and persists at enhancers of oncogenic regions in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a heterogeneous plasma cell malignancy that remains challenging to cure. Global hypomethylation correlates with an aggressive phenotype of the disease, while hypermethylation is observed at particular regions of myeloma such as B cell-specific enhancers. The recently discovered active epigenetic mark 5-hydroxymethylCytosine (5hmC) may also play a role in tumor biology; however, little is known about its level and distribution in myeloma. In this study, we investigated the global level and the genomic localization of 5hmC in myeloma cells from 40 newly diagnosed patients, including paired relapses, and of control individuals. RESULTS: Compared to normal plasma cells, we found global 5hmC levels to be lower in myeloma (P < 0.001). Higher levels of 5hmC were found in lower grades of the International Staging System prognostic index (P < 0.05) and tend to associate with a longer overall survival (P < 0.1). From the hydroxymethylome data, we observed that the remaining 5hmC is organized in large domains overlapping with active chromatin marks and chromatin opening. We discovered that 5hmC strongly persists at key oncogenic genes such as CCND1, CCND2 and MMSET and characterized domains that are specifically hydroxymethylated in myeloma subgroups. Novel 5hmC-enriched domains were found at putative enhancers of CCND2 and MYC in newly diagnosed patients. CONCLUSIONS: 5hmC level is associated with clinical aspects of MM. Mapping 5hmC at a genome-wide level provides insights into the disease biology directly from genomic DNA, which makes it a potent mark to study epigenetics on large patient cohorts.