Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.Singapore-MIT Alliance for Research and TechnologyNational Institute of Environmental Health Sciences (ES017010)National Institute of Environmental Health Sciences (ES002109)Singapore-MIT Alliance (Graduate Fellowship)Singapore. National Research Foundatio

The DNA-damage signature in Saccharomyces cerevisiae is associated with single-strand breaks in DNA

BACKGROUND: Upon exposure to agents that damage DNA, Saccharomyces cerevisiae undergo widespread reprogramming of gene expression. Such a vast response may be due not only to damage to DNA but also damage to proteins, RNA, and lipids. Here the transcriptional response of S. cerevisiae specifically induced by DNA damage was discerned by exposing S. cerevisiae to a panel of three "radiomimetic" enediyne antibiotics (calicheamicin γ(1)(I), esperamicin A1 and neocarzinostatin) that bind specifically to DNA and generate varying proportions of single- and double-strand DNA breaks. The genome-wide responses were compared to those induced by the non-selective oxidant γ-radiation. RESULTS: Given well-controlled exposures that resulted in similar and minimal cell death (~20–25%) across all conditions, the extent of gene expression modulation was markedly different depending on treatment with the enediynes or γ-radiation. Exposure to γ-radiation resulted in more extensive transcriptional changes classified both by the number of genes modulated and the magnitude of change. Common biological responses were identified between the enediynes and γ-radiation, with the induction of DNA repair and stress response genes, and the repression of ribosomal biogenesis genes. Despite these common responses, a fraction of the response induced by gamma radiation was repressed by the enediynes and vise versa, suggesting that the enediyne response is not entirely "radiomimetic." Regression analysis identified 55 transcripts with gene expression induction associated both with double- or single-strand break formation. The S. cerevisiae "DNA damage signature" genes as defined by Gasch et al. [1] were enriched among regulated transcripts associated with single-strand breaks, while genes involved in cell cycle regulation were associated with double-strand breaks. CONCLUSION: Dissection of the transcriptional response in yeast that is specifically signaled by DNA strand breaks has identified that single-strand breaks provide the signal for activation of transcripts encoding proteins involved in the DNA damage signature in S. cerevisiae, and double-strand breaks signal changes in cell cycle regulation genes

A Quantitative Systems Approach Reveals Dynamic Control of tRNA Modifications during Cellular Stress

Decades of study have revealed more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA, yet the larger functional dynamics of this conserved system are unclear. To this end, we developed a highly precise mass spectrometric method to quantify tRNA modifications in Saccharomyces cerevisiae. Our approach revealed several novel biosynthetic pathways for RNA modifications and led to the discovery of signature changes in the spectrum of tRNA modifications in the damage response to mechanistically different toxicants. This is illustrated with the RNA modifications Cm, m[superscript 5]C, and m[superscript 2][subscript 2]G, which increase following hydrogen peroxide exposure but decrease or are unaffected by exposure to methylmethane sulfonate, arsenite, and hypochlorite. Cytotoxic hypersensitivity to hydrogen peroxide is conferred by loss of enzymes catalyzing the formation of Cm, m[superscript 5]C, and m[superscript 2][subscript 2]G, which demonstrates that tRNA modifications are critical features of the cellular stress response. The results of our study support a general model of dynamic control of tRNA modifications in cellular response pathways and add to the growing repertoire of mechanisms controlling translational responses in cells.National Institute of Environmental Health Sciences (ES002109)National Institute of Environmental Health Sciences (ES017010)National Institute of Environmental Health Sciences (ES015037)National Cancer Institute (U.S.) (CA026731)National Center for Research Resources (U.S.) (RR023783)Singapore-MIT Alliance for Research and Technolog

Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella

Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction–modification (R–M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R–M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB–E, while restriction involves additional three genes dptF–H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R–M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification.National Natural Science Foundation (China) (Grant 31170085)National Natural Science Foundation (China) (Grant 31070058)Ministry of Science and Technology of the People's Republic of China (973 and 863 Programs)China Scholarship CouncilNational Science Foundation (U.S.) (Grant CHE-1019990)Shanghai Municipal Council of Science and Technology. Shanghai Pujiang Program (Grant 12PJD021

Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNA[superscript Met(CAU)] and tRNA[superscript Trp(CCA)] are substrates for Cm formation, tRNA[superscript Gln(UUG)], tRNA[superscript Pro(UGG)], tRNA[superscript Pro(CGG)] and tRNA[superscript His(GUG)] for Um, and tRNA[superscript Pro(GGG)] for Am. tRNA[superscript Ser(UGA)], previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNA[superscriptPro(GGG)] and Um in tRNA[superscript Gln(UUG)] by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.National Science Foundation (U.S.) (CHE-1308839)Agilent TechnologiesSingapore-MIT Alliance for Research and Technology (SMART

Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences

Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of G[subscript ps]AAC/G[subscript ps]TTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at C[subscript ps]CA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.National Natural Science Foundation (China)Ministry of Science and Technology of the People's Republic of China (973 and 863 Programs)Shanghai Municipal Council of Science and Technology. Shanghai Pujiang ProgramNational Science Foundation (U.S.) (CHE-1019990)National Institute of Environmental Health Sciences (ES002109)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

Current and emerging developments in subseasonal to decadal prediction

Weather and climate variations of subseasonal to decadal timescales can have enormous social, economic and environmental impacts, making skillful predictions on these timescales a valuable tool for decision makers. As such, there is a growing interest in the scientific, operational and applications communities in developing forecasts to improve our foreknowledge of extreme events. On subseasonal to seasonal (S2S) timescales, these include high-impact meteorological events such as tropical cyclones, extratropical storms, floods, droughts, and heat and cold waves. On seasonal to decadal (S2D) timescales, while the focus remains broadly similar (e.g., on precipitation, surface and upper ocean temperatures and their effects on the probabilities of high-impact meteorological events), understanding the roles of internal and externally-forced variability such as anthropogenic warming in forecasts also becomes important. The S2S and S2D communities share common scientific and technical challenges. These include forecast initialization and ensemble generation; initialization shock and drift; understanding the onset of model systematic errors; bias correct, calibration and forecast quality assessment; model resolution; atmosphere-ocean coupling; sources and expectations for predictability; and linking research, operational forecasting, and end user needs. In September 2018 a coordinated pair of international conferences, framed by the above challenges, was organized jointly by the World Climate Research Programme (WCRP) and the World Weather Research Prograame (WWRP). These conferences surveyed the state of S2S and S2D prediction, ongoing research, and future needs, providing an ideal basis for synthesizing current and emerging developments in these areas that promise to enhance future operational services. This article provides such a synthesis

The road less traveled: A new phosphorothioate antiviral defense mechanism discovered in Archaea

National Science Foundation (U.S.) (Grant CHE-1709364

Crystal structure and catalytic mechanism of the essential m¹ G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa

© 2019 Cold Spring Harbor Laboratory Press. All rights reserved. The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-L-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14± 3 μM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 μM) and uncompetitive for tRNA (Ki =6.4 ± 0.8 μM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.National Science Foundation (U.S.) (Grant CHE-1308839)Singapore-MIT Alliance. Innovation Centre (Grant ING137070-BIO)Thailand Research Fund (Grant 5980001