Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro

There are large differences between the cellular environment and the conditions widely used to study RNA structure and function in vitro, but until recently no technologies allowed nucleotide-resolution analyses of RNA structure inside cells. We have now examined the structure, dynamics, and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution using SHAPE. We compared the in-cell RNA structure with that obtained in aqueous buffer containing 1 mM Mg2+, approximately the concentration that we directly measured inside cells. The fully folded ligand-bound aptamer formed essentially the same structure in cells as in buffer. In contrast, the unbound RNA aptamer in cells was much more highly structured than the ligand-free state in vitro. Even high in vitro concentrations of Mg2+ did not yield the degree of structural organization observed for the free aptamer in cells. The crowded cellular environment thus stabilizes, or pre-organizes, otherwise dynamic RNA conformations significantly more than does Mg2+ alone, demonstrating a profound influence of the cellular environment on RNA structure. To deduce how the structural effects observed in cells may be induced in vitro and how the aptamer ligand binding affinity is affected by crowding, studies in the presence of total cellular RNA, and the synthetic polymer, polyethylene glycol (PEG), were employed. These studies showed that RNA-RNA interactions and PEG had varying effects on aptamer structure. The structure of the aptamer in the presence of both cellular RNA and large PEGs agreed better with the structure observed in cells. However, specific characteristics observed for the aptamer in cells were not observed in vitro under either condition. Crowding the aptamer with PEG had little effect on ligand binding affinity, even when large structural effects were observed. These results suggest that the complex cellular environment is difficult to mimic in vitro, and to do so, may require a combination of crowding agents having different chemical properties. Finally, the observed minimal effects on ligand binding affinity for the aptamer in the presence of PEG, suggests that while RNA structure is affected by crowding in cells, RNA function may not be significantly affected.Doctor of Philosoph

The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute timescale using SHAPE. The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg2+, the approximate Mg2+ concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg2+ concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg2+ alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations

Variation in hospital caesarean section rates for preterm births

Background: Evidence about optimal mode of delivery for preterm birth is lacking and there is thought to be considerable variation in practice. Objective: To assess whether variation in hospital preterm caesarean section rates (Robson Classification Group 10) and outcomes are explained by casemix, labour or hospital characteristics. Materials and Methods: Population-based cohort study in NSW, 2007-2011. Births were categorised according to degree of prematurity and hospital service capability: 26-31 weeks, 32-33 weeks and 34-36 weeks. Hospital preterm caesarean rates were investigated using multilevel logistic regression models, progressively adjusting for casemix, labour and hospital factors. The association between hospital caesarean rates, and severe maternal and neonatal morbidity rates was assessed. Results: At 26-31 weeks the caesarean rate was 55.2% (7 hospitals, range 43.4-58.4%); 50.9% at 32-33 weeks (12 hospitals, 43.4- 58.1%); and 36.4% at 34-36 weeks (51 hospitals, 17.4-48.3%). At 26-31 weeks and 32-33 weeks, 81% and 59% of the variation between hospitals was explained with no hospital significantly different from the state average after adjustment. At 34-36 weeks, although 59% of the variation was explained, substantial unexplained variation persisted. Hospital caesarean rates were not associated with severe maternal morbidity rates at any gestational age. At 26-31 weeks medium and high caesarean rates were associated with higher severe neonatal morbidity rates, but there was no evidence of this association ≥32 weeks. 3 Conclusion: Both casemix and practice differences contributed to the variation in hospital caesarean rates. Low preterm caesarean rates were not associated with worse outcomes.Australian National Health and Medical Research Council; Australian Research Counci

The Importance of Bcl-x L in the Survival of Human RPE Cells

PURPOSE: In normal eyes and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cell survival is critically important. Bcl-x(L) has been shown to be among the most highly expressed survival factors in cultured human RPE cells. In the current study the effect of Bcl-x(L) blockade on human RPE cell survival was determined under normal conditions and after induced oxidative stress. METHODS: Cultured human RPE cells from three different donors were transfected with modified, 2'-O-methoxyethoxy Bcl-x(L)-mismatched control antisense oligonucleotides (ASOs), Bcl-x(L)-specific ASOs, and Bcl-x(L) splice switching oligonucleotides (SSOs), which shift the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) into Bcl-x(S), a proapoptotic factor. RNA and protein were harvested at various time points after transfection. Bcl-x(L) and Bcl-x(S) mRNA transcript levels were analyzed using gene-specific primers with reverse transcription-polymerase chain reaction. Bcl-x(L) protein levels were analyzed using Western blot. Cell viability was measured by WST-1 and lactate dehydrogenase (LDH) assays. The mode of cell death was determined with a cell death ELISA and an M30 assay. To study the effects of oxidative stress, the cells were stimulated after transfection with various concentrations of H(2)O(2.) Cell viability was analyzed by WST-1 (Roche, Indianapolis, IN) and LDH assays RESULTS: After Bcl-x(L)-specific ASO and SSO transfections, Bcl-x(L) mRNA and protein levels were significantly reduced. Bcl-x(S) levels were increased after transfection with SSO. By day 8 after plating, the cells transfected with Bcl-x(L)-specific ASO had significantly decreased viability, which was further reduced by day 10. The SSO had an even more potent effect. Cell viability was reduced on day 4 after plating and by day 10, less than 10% of the cells were viable. Apoptotic cell death occurred as early as day 4 after plating. H(2)O(2), used as a model oxidant, further enhanced cell death induced by Bcl-x(L)-specific ASO and SSO. CONCLUSIONS: Bcl-x(L) plays an important role in human RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Treatment strategies that enhance Bcl-x(L) expression and/or prevent conversion of Bcl-x(L) to Bcl-x(S) may be useful in preventing RPE cell death in AMD. Treatments that reduce Bcl-x(L) and enhance Bcl-x(S) may be useful in inhibiting unwanted RPE cell proliferation in PVR

The Importance of Bcl-x L in the Survival of Human RPE Cells

PURPOSE. In normal eyes and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cell survival is critically important. Bcl-x L has been shown to be among the most highly expressed survival factors in cultured human RPE cells. In the current study the effect of Bcl-x L blockade on human RPE cell survival was determined under normal conditions and after induced oxidative stress. METHODS. Cultured human RPE cells from three different donors were transfected with modified, 2Ј-O-methoxyethoxy Bclx L -mismatched control antisense oligonucleotides (ASOs), Bclx L -specific ASOs, and Bcl-x L splice switching oligonucleotides (SSOs), which shift the splicing pattern of Bcl-x pre-mRNA from Bcl-x L into Bcl-x S , a proapoptotic factor. RNA and protein were harvested at various time points after transfection. Bcl-x L and Bcl-x S mRNA transcript levels were analyzed using genespecific primers with reverse transcription-polymerase chain reaction. Bcl-x L protein levels were analyzed using Western blot. Cell viability was measured by WST-1 and lactate dehydrogenase (LDH) assays. The mode of cell death was determined with a cell death ELISA and an M30 assay. To study the effects of oxidative stress, the cells were stimulated after transfection with various concentrations of H 2 O 2. Cell viability was analyzed by WST-1 (Roche, Indianapolis, IN) and LDH assays. RESULTS. After Bcl-x L -specific ASO and SSO transfections, Bcl-x L mRNA and protein levels were significantly reduced. Bcl-x S levels were increased after transfection with SSO. By day 8 after plating, the cells transfected with Bcl-x L -specific ASO had significantly decreased viability, which was further reduced by day 10. The SSO had an even more potent effect. Cell viability was reduced on day 4 after plating and by day 10, less than 10% of the cells were viable. Apoptotic cell death occurred as early as day 4 after plating. H 2 O 2 , used as a model oxidant, further enhanced cell death induced by Bcl-x L -specific ASO and SSO. CONCLUSIONS. Bcl-x L plays an important role in human RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Treatment strategies that enhance Bcl-x L expression and/or prevent conversion of Bcl-x L to Bcl-x S may be useful in preventing RPE cell death in AMD. Treatments that reduce Bcl-x L and enhance Bcl-x S may be useful in inhibiting unwanted RPE cell proliferation in PVR. (Invest Ophthalmol Vis Sci. 2007;48:3846 -3853) DOI:10.1167/iovs.06-1145 A ge-related macular degeneration (AMD) is the leading cause of irreversible blindness in adults over the age of 65 in the United States and currently affects more than 1.75 million individuals. 1 The exact etiology of AMD is unknown and is probably multifactorial. However, it has been hypothesized that cumulative oxidative stress throughout life and associated RPE cell injury play an important role. In normal eyes, there is very little RPE cell turnover, and most RPE cells survive for an individual's lifetime. In geographic atrophy, an advanced form of AMD, RPE cells die by apoptotic and nonapoptotic mechanisms. 2,3 RPE cell death is accompanied by underlying choriocapillaris atrophy and overlying retinal thinning, ultimately resulting in decreased visual acuity. In our laboratory, we have examined the effect of nuclear transcription factor (NF)-B inhibition on tumor necrosis factor (TNF)-␣-induced apoptosis in human RPE cells. 7 NF-B is a transcription factor that controls a wide range of genes, including genes that regulate apoptosis. 8 TNF-␣ is a cytokine that regulates a variety of RPE cell activities, including cell attachment, spreading, chemotaxis, migration, and proliferation

Challenge of Mimicking the Influences of the Cellular Environment on RNA Structure by PEG-Induced Macromolecular Crowding

There are large differences between the cellular environment and the conditions widely used to study RNA <i>in vitro</i>. SHAPE RNA structure probing in <i>Escherichia coli</i> cells has shown that the cellular environment stabilizes both long-range and local tertiary interactions in the adenine riboswitch aptamer domain. Synthetic crowding agents are widely used to understand the forces that stabilize RNA structure and in efforts to recapitulate the cellular environment under simplified experimental conditions. Here, we studied the structure and ligand binding ability of the adenine riboswitch in the presence of the macromolecular crowding agent, polyethylene glycol (PEG). Ethylene glycol and low-molecular mass PEGs destabilized RNA structure and caused the riboswitch to sample secondary structures different from those observed in simple buffered solutions or in cells. In the presence of larger PEGs, longer-range loop–loop interactions were more similar to those in cells than in buffer alone, consistent with prior work showing that larger PEGs stabilize compact RNA states. Ligand affinity was weakened by low-molecular mass PEGs but increased with high-molecular mass PEGs, indicating that PEG cosolvents exert complex chemical and steric effects on RNA structure. Regardless of polymer size, however, nucleotide-resolution structural characteristics observed in cells were not recapitulated in PEG solutions. Our results reveal that the cellular environment is difficult to recapitulate <i>in vitro</i>; mimicking the cellular state will likely require a combination of crowding agents and other chemical species

The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function <i>in vitro</i>. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing <i>Escherichia coli</i> cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg²⁺, the approximate Mg²⁺ concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg²⁺ concentrations to the buffer used for <i>in vitro</i> analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg²⁺ alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations

Discovery of (Dihydro)pyrazine <i>N</i>‑Oxides via Genome Mining in Pseudomonas

Overexpression of the Pseudomonas <i>virulence factor</i> (<i>pvf</i>) biosynthetic operon led to the identification of a family of pyrazine <i>N-</i>oxides (PNOs), including a novel dihydropyrazine <i>N,N</i>′-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)­PNOs was characterized, and a biosynthetic pathway for (d)­PNOs was proposed. This work highlights the unique chemistry catalyzed by <i>pvf</i>-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway