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BMC Oral Health Sep 2022Enterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence...
BACKGROUND
Enterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence factor of E. faecalis, extracellular polysaccharide (EPS) serves as a matrix to wrap bacteria and form biofilms. The homologous rnc gene, encoding Ribonuclease III, has been reported as a regulator of EPS synthesis. In order to develop novel anti-biofilm targets, we investigated the effects of the rnc gene on the biological characteristics of E. faecalis, and compared the biofilm tolerance towards the typical root canal irrigation agents and traditional Chinese medicine fluid Pudilan.
METHODS
E. faecalis rnc gene overexpression (rnc+) and low-expression (rnc-) strains were constructed. The growth curves of E. faecalis ATCC29212, rnc+, and rnc- strains were obtained to study the regulatory effect of the rnc gene on E. faecalis. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and crystal violet staining assays were performed to evaluate the morphology and composition of E. faecalis biofilms. Furthermore, the wild-type and mutant biofilms were treated with 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Pudilan. The residual viabilities of E. faecalis biofilms were evaluated using crystal violet staining and colony counting assays.
RESULTS
The results demonstrated that the rnc gene could promote bacterial growth and EPS synthesis, causing the EPS-barren biofilm morphology and low EPS/bacteria ratio. Both the rnc+ and rnc- biofilms showed increased susceptibility to the root canal irrigation agents. The 5% NaOCl group showed the highest biofilm removing effect followed by Pudilan and 2% CHX. The colony counting results showed almost complete removal of bacteria in the 5% NaOCl, 2% CHX, and Chinese medicine agents' groups.
CONCLUSIONS
This study concluded that the rnc gene could positively regulate bacterial proliferation, EPS synthesis, and biofilm formation in E. faecalis. The rnc mutation caused an increase in the disinfectant sensitivity of biofilm, indicating a potential anti-biofilm target. In addition, Pudilan exhibited an excellent ability to remove E. faecalis biofilm.
Topics: Chlorhexidine; Disinfectants; Disinfection; Enterococcus faecalis; Gentian Violet; Humans; Ribonuclease III; Sodium Hypochlorite; Virulence Factors
PubMed: 36127648
DOI: 10.1186/s12903-022-02462-1 -
Protein Science : a Publication of the... Jun 2021Dicer is a member of the ribonuclease III enzyme family and processes double-stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer...
Dicer is a member of the ribonuclease III enzyme family and processes double-stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non-canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double-stranded RNA-binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA-binding surface. The second dsRNA binding domain at C-terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem-loop structure of the RNA substrate, suggesting the possibility that stem-loop RNA-guided gene silencing pathway exists in budding yeast.
Topics: Fungal Proteins; Nucleic Acid Conformation; Protein Domains; Protein Multimerization; Protein Structure, Secondary; RNA, Fungal; Ribonuclease III; Saccharomycetales; Structure-Activity Relationship
PubMed: 33884665
DOI: 10.1002/pro.4086 -
Genes & Development Jun 2010MicroRNAs (miRNAs) modulate a broad range of gene expression patterns during development and tissue homeostasis, and in the pathogenesis of disease. The exquisite... (Review)
Review
MicroRNAs (miRNAs) modulate a broad range of gene expression patterns during development and tissue homeostasis, and in the pathogenesis of disease. The exquisite spatio-temporal control of miRNA abundance is made possible, in part, by regulation of the miRNA biogenesis pathway. In this review, we discuss two emerging paradigms for post-transcriptional control of miRNA expression. One paradigm centers on the Microprocessor, the protein complex essential for maturation of canonical miRNAs. The second paradigm is specific to miRNA families, and requires interaction between RNA-binding proteins and cis-regulatory sequences within miRNA precursor loops.
Topics: Animals; Gene Expression Regulation; Humans; MicroRNAs; RNA Precursors; RNA Processing, Post-Transcriptional; Ribonuclease III; Signal Transduction
PubMed: 20516194
DOI: 10.1101/gad.1919710 -
Molecular Cell May 2020A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood,...
A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.
Topics: Animals; Cryoelectron Microscopy; Humans; MicroRNAs; Models, Molecular; Protein Conformation; Protein Domains; RNA-Binding Proteins; Ribonuclease III; Spodoptera
PubMed: 32220645
DOI: 10.1016/j.molcel.2020.02.024 -
The New England Journal of Medicine Dec 2008
Topics: Biomarkers, Tumor; DEAD-box RNA Helicases; Endoribonucleases; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms; Prognosis; Ribonuclease III
PubMed: 19092157
DOI: 10.1056/NEJMe0808667 -
Nucleic Acids Research Apr 2011Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase...
Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ∼70-85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III.
Topics: Amino Acid Sequence; Bacteria; Base Pairing; Base Sequence; Biocatalysis; Cations, Divalent; Enzyme Stability; Glutamine; Hydrogen-Ion Concentration; Molecular Sequence Data; RNA Precursors; RNA, Bacterial; RNA, Double-Stranded; RNA, Ribosomal; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribonuclease III; Salts; Temperature
PubMed: 21138964
DOI: 10.1093/nar/gkq1030 -
FEBS Letters Oct 2005A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes.... (Review)
Review
A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.
Topics: Animals; Arabidopsis; Arabidopsis Proteins; Cell Cycle Proteins; DNA Methylation; Gene Expression Regulation, Plant; Genes, Plant; Genes, Reporter; Genome, Plant; Indoleacetic Acids; MicroRNAs; Models, Biological; Plant Leaves; Plant Physiological Phenomena; RNA, Plant; RNA, Small Interfering; Ribonuclease III; Signal Transduction
PubMed: 16144699
DOI: 10.1016/j.febslet.2005.07.071 -
Cell Death and Differentiation Oct 2012MicroRNAs (miRNAs) are non-coding RNAs that promote post-transcriptional silencing of genes involved in a wide range of developmental and pathological processes. It is... (Review)
Review
MicroRNAs (miRNAs) are non-coding RNAs that promote post-transcriptional silencing of genes involved in a wide range of developmental and pathological processes. It is estimated that most protein-coding genes harbor miRNA recognition sequences in their 3' untranslated region and are thus putative targets. While functions of miRNAs have been extensively characterized in various tissues, their multiple contributions to cerebral cortical development are just beginning to be unveiled. This review aims to outline the evidence collected to date demonstrating a role for miRNAs in cerebral corticogenesis with a particular emphasis on pathways that control the birth and maturation of functional excitatory projection neurons.
Topics: Animals; Cerebral Cortex; Humans; MicroRNAs; Models, Animal; Neurogenesis; Neuroglia; Ribonuclease III; Stem Cells
PubMed: 22858543
DOI: 10.1038/cdd.2012.96 -
Cell Jan 2016MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a...
MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
Topics: Amino Acid Sequence; Crystallography, X-Ray; DEAD-box RNA Helicases; Evolution, Molecular; Humans; MicroRNAs; Models, Chemical; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Protein Folding; Protein Structure, Tertiary; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Ribonuclease III; Sequence Alignment; Structural Homology, Protein
PubMed: 26748718
DOI: 10.1016/j.cell.2015.12.019 -
Cell Jun 2015MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional...
MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
Topics: Base Sequence; Dimerization; Humans; MicroRNAs; Molecular Sequence Data; Nucleotide Motifs; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Recombinant Proteins; Ribonuclease III
PubMed: 26027739
DOI: 10.1016/j.cell.2015.05.010