Ribozymes: the characteristics and properties of catalytic RNAs

Ribozymes, or catalytic RNAs, were discovered a little more than 15 years ago. They are found in the organelles of plants and lower eukaryotes, in amphibians, in prokaryotes, in bacteriophages, and in viroids and satellite viruses that infect plants. An example is also known of a ribozyme in hepatitis delta virus, a serious human pathogen. Additional ribozymes are bound to be found in the future, and it is tempting to regard the RNA component(s) of various ribonucleoprotein complexes as the catalytic engine, while the proteins serve as mere scaffolding - an unheard-of notion 15 years ago! In nature, ribozymes are involved in the processing of RNA precursors. However, all the characterized ribozymes have been converted, with some clever engineering, into RNA enzymes that can cleave or modify targeted RNAs (or even DNAs) without becoming altered themselves. While their success in vitro is unquestioned, ribozymes are increasingly used in vivo as valuable tools for studying and regulating gene expression. This review is intended as a brief introduction to the characteristics of the different identified ribozymes and their propertie

Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I (86AKTGSGKT93), motif III (228SAT230) and motif VI (375HRVGRTARG383). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI (383GTKGKGKS390). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNA

Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I ((86)AKTGSGKT(93)), motif III ((228)SAT(230)) and motif VI ((375)HRVGRTARG(383)). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI ((383)GTKGKGKS(390)). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNAs

Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers

International audienceDEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement

The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

International audienceThe DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrom-etry analyses, genetic experiments, sucrose gradients , in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs

Plant responses to fertilization experiments in lowland, species-rich, tropical forests.

We present a meta-analysis of plant responses to fertilization experiments conducted in lowland, species-rich, tropical forests. We also update a key result and present the first species-level analyses of tree growth rates for a 15-yr factorial nitrogen (N), phosphorus (P), and potassium (K) experiment conducted in central Panama. The update concerns community-level tree growth rates, which responded significantly to the addition of N and K together after 10 yr of fertilization but not after 15 yr. Our experimental soils are infertile for the region, and species whose regional distributions are strongly associated with low soil P availability dominate the local tree flora. Under these circumstances, we expect muted responses to fertilization, and we predicted species associated with low-P soils would respond most slowly. The data did not support this prediction, species-level tree growth responses to P addition were unrelated to species-level soil P associations. The meta-analysis demonstrated that nutrient limitation is widespread in lowland tropical forests and evaluated two directional hypotheses concerning plant responses to N addition and to P addition. The meta-analysis supported the hypothesis that tree (or biomass) growth rate responses to fertilization are weaker in old growth forests and stronger in secondary forests, where rapid biomass accumulation provides a nutrient sink. The meta-analysis found no support for the long-standing hypothesis that plant responses are stronger for P addition and weaker for N addition. We do not advocate discarding the latter hypothesis. There are only 14 fertilization experiments from lowland, species-rich, tropical forests, 13 of the 14 experiments added nutrients for five or fewer years, and responses vary widely among experiments. Potential fertilization responses should be muted when the species present are well adapted to nutrient-poor soils, as is the case in our experiment, and when pest pressure increases with fertilization, as it does in our experiment. The statistical power and especially the duration of fertilization experiments conducted in old growth, tropical forests might be insufficient to detect the slow, modest growth responses that are to be expected

Are large clinical trials in orthopaedic trauma justified?

© 2018 The Author(s). Background: The objective of this analysis is to evaluate the necessity of large clinical trials using FLOW trial data. Methods: The FLOW pilot study and definitive trial were factorial trials evaluating the effect of different irrigation solutions and pressures on re-operation. To explore treatment effects over time, we analyzed data from the pilot and definitive trial in increments of 250 patients until the final sample size of 2447 patients was reached. At each increment we calculated the relative risk (RR) and associated 95% confidence interval (CI) for the treatment effect, and compared the results that would have been reported at the smaller enrolments with those seen in the final, adequately powered study. Results: The pilot study analysis of 89 patients and initial incremental enrolments in the FLOW definitive trial favored low pressure compared to high pressure (RR: 1.50, 95% CI: 0.75-3.04; RR: 1.39, 95% CI: 0.60-3.23, respectively), which is in contradiction to the final enrolment, which found no difference between high and low pressure (RR: 1.04, 95% CI: 0.81-1.33). In the soap versus saline comparison, the FLOW pilot study suggested that re-operation rate was similar in both the soap and saline groups (RR: 0.98, 95% CI: 0.50-1.92), whereas the FLOW definitive trial found that the re-operation rate was higher in the soap treatment arm (RR: 1.28, 95% CI: 1.04-1.57). Conclusions: Our findings suggest that studies with smaller sample sizes would have led to erroneous conclusions in the management of open fracture wounds. Trial registration: NCT01069315 (FLOW Pilot Study) Date of Registration: February 17, 2010, NCT00788398 (FLOW Definitive Trial) Date of Registration: November 10, 2008

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery