Bioinformatic prediction of proteins relevant to functions of the bacterial OLE ribonucleoprotein complex.

OLE (ornate, large, extremophilic) RNAs are members of a noncoding RNA class present in many Gram-positive, extremophilic bacteria. The large size, complex structure, and extensive sequence conservation of OLE RNAs are characteristics consistent with the hypothesis that they likely function as ribozymes. The OLE RNA representative from Halalkalibacterium halodurans is known to localize to the phospholipid membrane and requires at least three essential protein partners: OapA, OapB, and OapC. However, the precise biochemical functions of this unusual ribonucleoprotein (RNP) complex remain unknown. Genetic disruption of OLE RNA or its partners revealed that the complex is beneficial under diverse stress conditions. To search for additional links between OLE RNA and other cellular components, we used phylogenetic profiling to identify proteins that are either correlated or anticorrelated with the presence of OLE RNA in various bacterial species. This analysis revealed strong correlations between the essential protein-binding partners of OLE RNA and organisms that carry the ole gene. Similarly, proteins involved in sporulation are correlated, suggesting a potential role for the OLE RNP complex in spore formation. Intriguingly, the Mg2+ transporter MpfA is strongly anticorrelated with OLE RNA. Evidence indicates that MpfA is structurally related to OapA and therefore MpfA may serve as a functional replacement for some contributions otherwise performed by the OLE RNP complex in species that lack this device. Indeed, OLE RNAs might represent an ancient RNA class that enabled primitive organisms to sense and respond to major cellular stresses.IMPORTANCEOLE (ornate, large, extremophilic) RNAs were first reported nearly 20 years ago, and they represent one of the largest and most intricately folded noncoding RNA classes whose biochemical function remains to be established. Other RNAs with similar size, structural complexity, and extent of sequence conservation have proven to catalyze chemical transformations. Therefore, we speculate that OLE RNAs likewise operate as ribozymes and that they might catalyze a fundamental reaction that has persisted since the RNA World era-a time before the emergence of proteins in evolution. To seek additional clues regarding the function of OLE RNA, we undertook a computational effort to identify potential protein components of the OLE ribonucleoprotein (RNP) complex or other proteins that have functional links to this device. This analysis revealed known protein partners and several additional proteins that might be physically or functionally linked to the OLE RNP complex. Finally, we identified a Mg2+ transporter protein, MpfA, that strongly anticorrelates with the OLE RNP complex. This latter result suggests that MpfA might perform at least some functions that are like those carried out by the OLE RNP complex.

Disruption of the bacterial OLE RNP complex impairs growth on alternative carbon sources.

Ornate, large, extremophilic (OLE) RNAs comprise a class of large noncoding RNAs in bacteria whose members form a membrane-associated ribonucleoprotein (RNP) complex. This complex facilitates cellular adaptation to diverse stresses such as exposure to cold, short-chain alcohols, and elevated Mg2+ concentrations. Here, we report additional phenotypes exhibited by Halalkalibacterium halodurans (formerly called Bacillus halodurans) strains lacking functional OLE RNP complexes. Genetic disruption of the complex causes restricted growth compared to wild-type cells when cultured in minimal media (MM) wherein glucose is replaced with alternative carbon/energy sources. Genetic suppressor selections conducted in glutamate MM yielded isolates that carry mutations in or near genes relevant to Mn2+ homeostasis (ykoY and mntB), phosphate homeostasis (phoR), and putative multidrug resistance (bmrCD). These functional links between OLE RNA, carbon/energy management, and other fundamental processes including protein secretion are consistent with the hypothesis that the OLE RNP complex is a major contributor to cellular adaptation to unfavorable growth conditions.

Genetic disruption of the bacterial raiA motif noncoding RNA causes defects in sporulation and aggregation.

Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the "raiA motif" has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor.

Experience of primary caregivers in utilising an mHealth application for remote dental screening in preschool children.

Objectives This study aimed to address the acceptance of mHealth applications for a dental screening app that facilitates patient information entry and captures dental photos remotely to assist in caries diagnosis in preschool children in Australia. Methods All participants were recruited through the ORIGINS Project, a community-based interventional birth cohort study in Western Australia. Forty-two primary caregivers, who were the users of a teledental screening app, were given a questionnaire with 17 questions; these were constructed based on the theme of the Technology Acceptance Model: perceived ease of use (PE), perceived usefulness (PU), behavioural intention to adopt (BI), anxiety (ANX), attitude toward a behaviour (ATB), and self-efficacy (SE). Cronbach's alpha was estimated to determine internal consistency. Path analysis was employed to quantify the relationship between each theme. Results The mean values for most themes indicated high satisfaction with the intervention among caregivers (scores out of 5): PE (4.54 ± 0.55), PU (4.65 ± 0.49), BI (4.40 ± 0.65), ATB (4.23 ± 0.70), SE (4.36 ± 0.64). Results indicated high consistency in response in the PE, PU, ATB, and SE (α = 0.74-0.84) and moderate consistency was observed in ANX and BI (α = 0.50-0.62). The overall intention of using the dental screening app was significantly related to both PU and ATB (P Conclusion The perceived usefulness and attitude toward behaviours influenced the overall behavioural intention of the participants to use the telehealth model in dental screening. Recognising these relationships indicates community readiness for implementing the telehealth application in the dental program and enables identification of areas for improving its diffusion.

Evidence that OLE RNA is a component of a major stress-responsive ribonucleoprotein particle in extremophilic bacteria.

OLE RNA is a ~600-nucleotide noncoding RNA present in many Gram-positive bacteria that thrive mostly in extreme environments, including elevated temperature, salt, and pH conditions. The precise biochemical functions of this highly conserved RNA remain unknown, but it forms a ribonucleoprotein (RNP) complex that localizes to cell membranes. Genetic disruption of the RNA or its essential protein partners causes reduced cell growth under various stress conditions. These phenotypes include sensitivity to short-chain alcohols, cold intolerance, reduced growth on sub-optimal carbon sources, and intolerance of even modest concentrations of Mg2+ . Thus, many bacterial species appear to employ OLE RNA as a component of an intricate RNP apparatus to monitor fundamental cellular processes and make physiological and metabolic adaptations. Herein we hypothesize that the OLE RNP complex is functionally equivalent to the eukaryotic TOR complexes, which integrate signals from various diverse pathways to coordinate processes central to cell growth, replication, and survival.

The balancing act of R-loop biology: The good, the bad, and the ugly.

An R-loop is a three-stranded nucleic acid structure that consists of a DNA:RNA hybrid and a displaced strand of DNA. R-loops occur frequently in genomes and have significant physiological importance. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Several studies have uncovered that R-loops contribute to fundamental biological processes in various organisms. Paradoxically, although they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability. Recent evidence suggests that R-loops are involved in a number of human diseases, including neurological disorders, cancer, and autoimmune diseases. This review focuses on the molecular basis for R-loop-mediated gene regulation and genomic instability and briefly discusses methods for identifying R-loops in vivo It also highlights recent studies indicating the role of R-loops in DNA double-strand break repair with an updated view of much-needed future goals in R-loop biology.