Small molecule control of pre-mRNA splicing

SR proteins regulate alternative splicing by binding to exonic sequences where, via an arginine/serine-rich splicing activation domain, they enhance the binding of the spliceosome to the adjacent splice sites. Here, a system is described in which a nontoxic derivative of the small molecule rapamycin is used to control pre-mRNA splicing in vitro. This involves the rapamycin-dependent recruitment of a splicing activation domain located on one protein to a second protein bound to the pre-mRNA. These results provide a new approach to explore for regulating gene expression in vivo with small molecules by controlling pre-mRNA splicing.     

Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures

Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons--the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA.     

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.     

Microvillar components of light adaptation in blowflies

The process of light adaptation in blowfly photoreceptors was analyzed using intracellular recording techniques and double and triple flash stimuli. Adapting flashes of increasing intensity caused a progressive reduction in the excitability of the photoreceptors, which became temporarily suppressed when 3 x 10(6) quanta were absorbed by the cell. This suppression was confirmed by subsequently applying an intense test flash that photoactivated a considerable fraction of the 10(8) visual pigment molecules in the cell. The period of temporary desensitization is referred to as the refractory period. The stimulus intensity to render the receptor cell refractory was found to be independent of the extracellular calcium concentration over a range of 10(-4) and 10(-2) M. During the refractory period (30-40 ms after the adapting flash) the cell appears to be "protected" against further light adaptation since light absorption during this period did not affect the recovery of the cell's excitability. Calculations showed that the number of quantum absorptions necessary to induce receptor refractoriness is just sufficient to photoactivate every microvillus of the rhabdomere. This coincidence led to the hypothesis that the refractoriness of the receptor cells is due to the refractoriness of the individual microvilli. The sensitivity of the receptor cells after relatively weak adapting flashes was reduced considerably more than could be accounted for by the microvilli becoming refractory. A quantitative analysis of these results suggests that a photoactivated microvillus induces a local adaptation over a relatively small area of the rhabdomere around it, which includes several tens of microvilli. After light adaptation with an intense flash, photoactivation of every microvillus by the absorption of a few quanta produced only a small receptor response whereas photoactivation of every rhodopsin molecule in every microvillus produced the maximum response. The excitatory efficiency of the microvilli therefore increases with the number of quanta that are absorbed simultaneously.     

Effects of recent increases in salinity and nutrient concentrations on the microbialite community of Lake Clifton (Western Australia): are the thrombolites at risk?

There has been a strong research focus on optical properties in temperate estuaries but very much less in tropical estuaries. These properties comprise light and beam attenuation dominated by suspended particulate matter, Chl a, and CDOM. Spatially and temporally distributed data on optical properties in a tropical wet and dry estuary are compared and discussed in relation to those of temperate estuaries. Sampling in the Nha Phu estuary, Vietnam, consisted of five stations on a transect from head to mouth that was sampled four times during dry conditions and three times during wet conditions between May 2006 and April 2008. Methods comprised CTD, optical measurements, and water sampling for suspended matter, Chl a, and CDOM. Results showed high light attenuation—K _d(PAR)—in wet conditions and low in dry. K _d(PAR) was highest at the estuary head and lower in the outer part. Spatial and temporal variations in K _d(PAR) were in general dominated by variations in suspended particulate matter concentrations in both wet and dry conditions. Chl a concentrations were low and showed no strong variations between wet and dry conditions. CDOM absorption coefficients were higher in wet conditions with high values at the head and lower in the central part of the estuary. The depth of the photic zone was reduced by up to 50% during wet conditions. A residence time in the estuary of 5–6 days was derived from the rate of change of K _d(PAR) after a period of heavy rain and discharge of freshwater into the estuary. This complied with a residence time of four and a half days derived from a basic physical relation. Optical properties were in general comparable to temperate estuaries in dry conditions although Chl a concentrations were lower in Nha Phu. A second distinctive point, as compared to temperate estuaries, was the episodic character with days of strong rainfall followed by longer periods of dry weather. All sampling, both wet and dry, was carried out in the dry season which implies a less definitive perception of wet and dry seasons.     

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.     

Kelp and sea urchin settlement mediated by biotic interactions with benthic coralline algal species

Species interactions can influence key ecological processes that support community assembly and composition. For example, coralline algae encompass extensive diversity and may play a major role in regime shifts from kelp forests to urchin-dominated barrens through their role in inducing invertebrate larval metamorphosis and influencing kelp spore settlement. In a series of laboratory experiments, we tested the hypothesis that different coralline communities facilitate the maintenance of either ecosystem state by either promoting or inhibiting early recruitment of kelps or urchins. Coralline algae significantly increased red urchin metamorphosis compared with a control, while they had varying effects on kelp settlement. Urchin metamorphosis and density of juvenile canopy kelps did not differ significantly across coralline species abundant in both kelp forests and urchin barrens, suggesting that recruitment of urchin and canopy kelps does not depend on specific corallines. Non-calcified fleshy red algal crusts promoted the highest mean urchin metamorphosis percentage and showed some of the lowest canopy kelp settlement. In contrast, settlement of one subcanopy kelp species was reduced on crustose corallines, but elevated on articulated corallines, suggesting that articulated corallines, typically absent in urchin barrens, may need to recover before this subcanopy kelp could return. Coralline species differed in surface bacterial microbiome composition; however, urchin metamorphosis was not significantly different when microbiomes were removed with antibiotics. Our results clarify the role played by coralline algal species in kelp forest community assembly and could have important implications for kelp forest recovery.