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Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2'O-methylation

ABSTRACT: TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 inTLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2'O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8.

SUBMITTER: Mrs. Ivanéia, V Nunes

PROVIDER: S-SCDT-10_1038-S44319-024-00281-9 | biostudies-other |

REPOSITORIES: biostudies-other

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Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2’O-methylation

Project description:Mechanistic studies have revealed that TLR8 senses single-stranded RNA (ssRNA) fragments, processed via synergistic cleavage by ribonucleases (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to recognition of bacterial RNA by TLR8. Herein, we characterized molecular RNase 6 cleavage mechanisms. BLaER1 RNASE6-/- cells showed a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) but also upon infection with live whole bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generated fragments that induced stimulation in RNASE6 knockout cells. 2’O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impaired upstream processing by RNase 6 and dampened TLR8 stimulation. In summary, this data shows that RNase 6 plays a critical role in the processing of bacterial RNA by generating uridine-terminated breakdown products that ultimately activate TLR8.

2024-09-12 | PXD053881 | Pride

Coevolution of RNA and protein subunits in RNase P and RNase MRP, two RNA processing enzymes.

Project description:RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.

| S-EPMC10966300 | biostudies-literature

Stable transgenerational epigenetic inheritance requires a DNA methylation-sensing circuit.

Project description:Epigenetic states are stably propagated in eukaryotes. In plants, DNA methylation patterns are faithfully inherited over many generations but it is unknown how the dynamic activities of cytosine DNA methyltransferases and 5-methylcytosine DNA glycosylases interact to maintain epigenetic homeostasis. Here we show that a methylation-sensing gene regulatory circuit centered on a 5-methylcytosine DNA glycosylase gene is required for long-term epigenetic fidelity in Arabidopsis. Disrupting this circuit causes widespread methylation losses and abnormal phenotypes that progressively worsen over generations. In heterochromatin, these losses are counteracted such that methylation returns to a normal level over four generations. However, thousands of loci in euchromatin progressively lose DNA methylation between generations and remain unmethylated. We conclude that an actively maintained equilibrium between methylation and demethylation activities is required to ensure long-term stable inheritance of epigenetic information.

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Structural basis of RNA processing by human mitochondrial RNase P.

Project description:Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.

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RNase MRP and the RNA processing cascade in the eukaryotic ancestor.

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Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Project description:Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

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Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation.

Project description:RNA 2'-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3'-5' exoribonuclease and is particularly sensitive to RNA 2'-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2'-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR's ribonuclease activity and significant sensitivity to RNA 2'-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2'-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2'-O-methylations research in biological and clinical samples.

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Information processing and signal integration in bacterial quorum sensing.

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Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

Project description:MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation.

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A bacterial methyltransferase M.EcoHK311 requires two proteins for in vitro methylation.

Project description:The genes encoding EcoHK311 restriction-modification (R-M) system were isolated from a clinically-isolated Escherichia coli strain HK31. The entire R-M system of EcoHK311 is located in a 2.1 kb fragment. R.EcoHK311 is an isoschizomer of Eael which recognizes and cleaves Y decreases GGCCR. M.EcoHK31l consists of two polypeptides alpha and beta with sizes 309 and 176 aa, respectively. Polypeptide beta is encoded within aa, alternative reading frame of polypeptide alpha. All the conserved motifs in mC5-MTases can be found in polypeptide alpha except motif IX which is present in polypeptide beta. Polypeptides alpha and beta were separately synthesized in a T7 promoter controlled over-expression system and in vitro methylation occurred only when the two extracts were mixed and thus confirms that two polypeptides are required for methylation.

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