Goldilocks and RNA: where Mg2+ concentration is just right.

Magnesium, the most abundant divalent cation in cells, catalyzes RNA cleavage but also promotes RNA folding. Because folding can protect RNA from cleavage, we predicted a 'Goldilocks landscape', with local maximum in RNA lifetime at Mg2+ concentrations required for folding. Here, we use simulation and experiment to discover an innate and sophisticated mechanism of control of RNA lifetime. By simulation we characterized RNA Goldilocks landscapes and their dependence on cleavage and folding parameters. Experiments with yeast tRNAPhe and the Tetrahymena ribozyme P4-P6 domain show that structured RNAs can inhabit Goldilocks peaks. The Goldilocks peaks are tunable by differences in folded and unfolded cleavage rate constants, Mg2+ binding cooperativity, and Mg2+ affinity. Different folding and cleavage parameters produce Goldilocks landscapes with a variety of features. Goldilocks behavior allows ultrafine control of RNA chemical lifetime, whereas non-folding RNAs do not display Goldilocks peaks of protection. In sum, the effects of Mg2+ on RNA persistence are expected to be pleomorphic, both protecting and degrading RNA. In evolutionary context, Goldilocks behavior may have been a selectable trait of RNA in an early Earth environment containing Mg2+ and other metals.

Human ribosomal G-quadruplexes regulate heme bioavailability.

The in vitro formation of stable G-quadruplexes (G4s) in human rRNA was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme-affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest that ribosomes play a role in regulating heme homeostasis.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+.

UNLABELLED: Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. CONCLUSION: Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.

Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection.

UNLABELLED: Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGFbeta1). TGFbeta1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFbeta1. CONCLUSION: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis.

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

G-Quadruplexes in Human Ribosomal RNA.

rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans. Here we show that the terminus of an rRNA tentacle of Homo sapiens contains 10 tandem G-tracts that form highly stable G-quadruplexes in vitro. We characterized rRNA of the H. sapiens large ribosomal subunit by computation, circular dichroism, UV melting, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts, and blotting. We investigated Expansion Segment 7 (ES7), oligomers derived from ES7, intact 28S rRNA, 80S ribosomes, and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. These proteins include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins. Finally, by multiple sequence alignments, we observe that G-quadruplex-forming sequences are a general feature of LSU rRNA of Chordata but not, as far as we can tell, of other species. Chordata ribosomes present polymorphic tentacles with the potential to switch between inter- and intramolecular G-quadruplexes. To our knowledge, G-quadruplexes have not been reported previously in ribosomes.