Strong Eukaryotic IRESs Have Weak Secondary Structure

BACKGROUND: The objective of this work was to investigate the hypothesis that eukaryotic Internal Ribosome Entry Sites (IRES) lack secondary structure and to examine the generality of the hypothesis. METHODOLOGY/PRINCIPAL FINDINGS: IRESs of the yeast and the fruit fly are located in the 5'UTR immediately upstream of the initiation codon. The minimum folding energy (MFE) of 60 nt RNA segments immediately upstream of the initiation codons was calculated as a proxy of secondary structure stability. MFE of the reverse complements of these 60 nt segments was also calculated. The relationship between MFE and empirically determined IRES activity was investigated to test the hypothesis that strong IRES activity is associated with weak secondary structure. We show that IRES activity in the yeast and the fruit fly correlates strongly with the structural stability, with highest IRES activity found in RNA segments that exhibit the weakest secondary structure. CONCLUSIONS: We found that a subset of eukaryotic IRESs exhibits very low secondary structure in the 5'-UTR sequences immediately upstream of the initiation codon. The consistency in results between the yeast and the fruit fly suggests a possible shared mechanism of cap-independent translation initiation that relies on an unstructured RNA segment

Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation

Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin ?3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered

Systematic monitoring of heathy woodlands in a Mediterranean climate - a practical assessment of methods

Practical and useful vegetation monitoring methods are needed, and data compatibility and validation of remotely sensed data are desirable. Methods have not been adequately tested for heathy woodlands. We tested the feasibility of detecting species composition shifts in remnant woodland in South Australia, comparing historical (1986) plot data with temporal replicates (2010). We compared the uniformity of species composition among spatially scattered versus spatially clustered plots. At two sites, we compared visual and point-intercept estimation of cover and species diversity. Species composition (presence/absence) shifted between 1986 and 2010. Species that significantly shifted in frequency had low cover. Observations of decreasing species were consistent with predictions from temperature response curves (generalised additive models) for climate change over the period. However, long-term trends could not be distinguished from medium-term dynamics or short-term changes in visibility from this dataset. Difficulties were highlighted in assessing compositional change using historical baselines established for a different purpose in terms of spatial sampling and accuracy of replicate plots, differences in standard plot methods and verification of species identifications. Spatially clustered replicate plots were more similar in species composition than spatially scattered plots, improving change detection potential but decreasing area of inference. Visual surveys detected more species than point-intercepts. Visual cover estimates differed little from point-intercepts although underestimating cover in some instances relative to intercepts. Point-intercepts provide more precise cover estimates of dominant species but took longer and were difficult in steep, heathy terrain. A decision tree based on costs and benefits is presented assessing monitoring options based on data presented. The appropriate method is a function of available resources, the need for precise cover estimates versus adequate species detection, replication and practical considerations such as access and terrain.Greg R. Guerin & Andrew J. Low

Snowball earth termination by destabilization of equatorial permafrost methane clathrate

Martin Kennedy, David Mrofka and Chris von der Borc

The effect of present day in situ stresses and paleo-stresses on locating sweet spots in unconventional reservoirs, a case study from Moomba-Big Lake fields, Cooper Basin, South Australia

The effect of stresses on permeability is a combination of external stress and pore pressure. We are examining if and how present-day in situ stresses and the spatial distribution of permeable domains in the Moomba-Big Lake fields in the Cooper Basin are correlated. We analysed image logs, well logs, and formation tests and calculated the orientation and magnitudes of the three principal stresses. A 3-dimensional model was constructed and the calculated stress magnitudes and orientations were applied to the model. The resulting stress distribution under the current day stress state showed a highly permeable domain indicating a sweet spot in the Big Lake field. This is currently the location of a gas pool that forms, with the Moomba field, one-third of the gas reserve in SA. No potential sweet spots are located in the Moomba area according to the stress model. We also used the finite element method (FEM) and the boundary element method (BEM) for modelling the behaviour of folds, fractures, and faults that formed during the tectonic history of the basin. We used geomechanical restoration techniques for locating sweet spots in the Moomba-Big Lake fields. The methodology attempts to reconstruct the current day structural and geometrical placement and predicts fractures generated due to stresses released during past tectonic events. Orientation of predicted fractures using FEM-based geomechanical restoration correlated well with the orientation of the image log fractures. The spatial distribution of paleo-stresses applied on the predicted fractures showed a potentially stressed fracture set in the location of the currently producing Big Lake sweet spot. However, orientation of predicted fractures using BEM-based geomechanical restoration correlated well next to the Big Lake fault but did not show any correlation away from the major fault. This is due to the fact that BEM restoration takes in consideration fault dislocation as the only driver of fracture generation and ignores the other factors. However, paleo-stress distribution using BEM restoration predicted the same producing area but with less accuracy due to the fundamentals of the BEM. No fracture density information can be extracted from any of the methods as the methodologies generate fractures with density that depends on the initial project mesh size. Accordingly, these methodologies can be used for locating the current-day and paleo-stresses, as well as fracture orientation but not density. Also, reservoir permeability is proved in this study to be controlled by a combination of current day and stored paleo-stresses.H. Abul Khair, D. Cooke, M. Han