RNA structure and the mechanisms of alternative splicing

Alternative splicing is a widespread means of increasing protein diversity and regulating gene expression in eukaryotes. Much progress has been made in understanding the proteins involved in regulating alternative splicing, the sequences they bind to, and how these interactions lead to changes in splicing patterns. However, several recent studies have identified other players involved in regulating alternative splicing. A major theme emerging from these studies is that RNA secondary structures play an under appreciated role in the regulation of alternative splicing. This review provides an overview of the basic aspects of splicing regulation and highlights recent progress in understanding the role of RNA secondary structure in this process.     

Genetically Modified Ruminal Bacteria Protect Sheep from Fluoroacetate Poisoning

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10⁶ to 10⁷ cells ml⁻¹ in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.     

Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI-MS/MS

Ginkgo biloba is one of the most popular herb nutrition supplements, with terpene lactones and flavonoids being the two major active components. A fingerprint profile method was developed using a capillary HPLC/MS method which can identify more than 70 components from the G. biloba product. The method allows the flavonoids and terpene lactones to be detected simultaneously and information of both the parent ion and its fragmentation can be obtained in just one HPLC/MS run. Targeted post-acquisition analysis allows mass spectrometric information regarding the identification of flavonoid components to be easily distinguished from other data, however the same approach for terpene lactones was less successful due to dimer formation and requires further development. The fingerprint profiles of five commercial G. biloba nutritional supplements were obtained and compared; variation of some components among the samples was observed and fortification could be detected. In the quality control analysis of the G. biloba product this method could be viewed as complementary to specific quantitative analysis of some bioactive components of the herb.     

Sixty years of genome biology

Sixty years after Watson and Crick published the double helix model of DNA''s structure, thirteen members of Genome Biology''s Editorial Board select key advances in the field of genome biology subsequent to that discovery.April 25th 2013 is the sixtieth anniversary of the infamous Watson and Crick Nature paper describing a model for the structure of DNA, published 25 April 1953: the now infamous ''double helix'' [1]. Two accompanying papers from Rosalind Franklin, Maurice Wilkins and colleagues leant experimental support to the proposed structure in the form of X-ray diffraction data [2,3], as described elsewhere in this issue of Genome Biology [4]. The model was a landmark discovery in the history of modern science, and was notable for its cross-disciplinary importance: the question addressed was of immense biological importance, but it was physicists and chemists whose expertise and techniques were needed in order to arrive at an answer. One of these physicists, Ray Gosling, describes the unveiling of Watson and Crick''s double helix structure as a ''eureka'' moment [4]: its simplicity and elegance were striking, and not only explained the X-ray diffraction data but also the mode of replication of life itself. It is rare for a scientific discovery to achieve such an iconic status, to pervade popular culture and the public consciousness, as well as to become an emblem of scientific inquiry - as exemplified by Genome Biology''s double helix-inspired logo. Although Avery had already shown DNA to be the genetic material [5], it took the convincing simplicity of Watson and Crick''s double helix for this notion to widely take hold, in place of theories favoring proteins. The discovery, therefore, had many important implications, and set the scene for future breakthroughs in the field of genome biology.To celebrate sixty years of such discoveries, we asked a jury composed of Genome Biology Editorial Board members to select key advances in the field since 25 April 1953. The brief was to choose a development that was either the most important or the most surprising, or that had the most personal impact, and to briefly summarize why. A number of selections focused on technological advances - from restriction mapping through microarrays and high-throughput sequencing. These technologies have clearly done much to inform our understanding of the biology of genomes. The most popular choice, however, was the discovery of introns. Much like the double helix, this discovery had something of the ''X factor'' to it: biologists trained in the post-intron era may take the concept of gene fragmentation for granted, but at the time it was a truly radical and paradigm-shifting idea. The sense of surprise made a strong impression on those old enough to remember the discovery, and one of the groups involved went so far as to describe it as ''amazing'' in the title of their paper [6].     

The role of F-actin and myosin in epithelial cell rheology

Although actin and myosin are important contributors to cell-force generation, shape change, and motility, their contributions to cell stiffness and frequency-dependent rheology have not been conclusively determined. We apply several pharmacological interventions to cultured epithelial cells to elucidate the roles of actin and myosin in the mechanical response of cells and intracellular fluctuations. A suite of different methods is used to separately examine the mechanics of the deep cell interior and cortex, in response to depletion of intracellular ATP, depolymerization of F-actin, and inhibition of myosin II. Comparison of these results shows that F-actin plays a significant role in the mechanics of the cortical region of epithelial cells, but its disruption has no discernable effect on the rheology of the deeper interior. Moreover, we find that myosins do not contribute significantly to the rheology or ATP-dependent, non-Brownian motion in the cell interior. Finally, we investigate the broad distribution of apparent stiffness values reported by some microrheology methods, which are not observed with two-point microrheology. Based on our findings and a simple model, we conclude that heterogeneity of the tracer-cytoskeleton contacts, rather than the network itself, can explain the broad distribution of apparent stiffnesses.     

Plant proteome analysis by mass spectrometry: principles, problems, pitfalls and recent developments

The genome of several species has now been elucidated; these genomes indicate the proteomic potential of the cell. While identification of genomes has been, and continues to be, a technically and intellectually demanding process, the identification of the proteome contains inherently greater difficulties. The proteome of each living cell is dynamic, altering in response to the individual cell's metabolic state and reception of intracellular and extracellular signal molecules, and many of the proteins which are expressed will be post-translationally altered. Thus if the purpose of the proteome analysis is to aid the understanding of protein function and interaction, then it is identification of the proteins in their final state which is required: for this mass spectrometric identification of individual proteins, indicating site and nature of modifications, is essential. Here we review the principles of the methodologies involved in such analyses, give some indication of current achievements in plant proteomics, and indicate imminent and prospective technical developments.     

CF45-1, a secreted protein which participates in Dictyostelium group size regulation

Developing Dictyostelium cells aggregate to form fruiting bodies containing typically 2 × 10⁴ cells. To prevent the formation of an excessively large fruiting body, streams of aggregating cells break up into groups if there are too many cells. The breakup is regulated by a secreted complex of polypeptides called counting factor (CF). Countin and CF50 are two of the components of CF. Disrupting the expression of either of these proteins results in cells secreting very little detectable CF activity, and as a result, aggregation streams remain intact and form large fruiting bodies, which invariably collapse. We find that disrupting the gene encoding a third protein present in crude CF, CF45-1, also results in the formation of large groups when cells are grown with bacteria on agar plates and then starve. However, unlike countin⁻ and cf50⁻ cells, cf45-1⁻ cells sometimes form smaller groups than wild-type cells when the cells are starved on filter pads. The predicted amino acid sequence of CF45-1 has some similarity to that of lysozyme, but recombinant CF45-1 has no detectable lysozyme activity. In the exudates from starved cells, CF45-1 is present in a ~450-kDa fraction that also contains countin and CF50, suggesting that it is part of a complex. Recombinant CF45-1 decreases group size in colonies of cf45-1⁻ cells with a 50% effective concentration (EC₅₀) of ~8 ng/ml and in colonies of wild-type and cf50⁻ cells with an EC₅₀ of ~40 ng/ml. Like countin⁻ and cf50⁻ cells, cf45-1⁻ cells have high levels of cytosolic glucose, high cell-cell adhesion, and low cell motility. Together, the data suggest that CF45-1 participates in group size regulation in Dictyostelium.