RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF⁶⁵) and small (hU2AF³⁵) subunits. hU2AF⁶⁵ contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF³⁵ has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF⁶⁵. While the requirement for the three RRMs on hU2AF⁶⁵ is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. Surprisingly, the RS domain on dU2AF³⁸ was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

Developing a functional connectivity indicator to detect change in fragmented landscapes

Biodiversity indicators are increasingly used to assess progress towards conservation targets. Particular indicators are required to assess the impacts of habitat fragmentation on landscape connectivity and biodiversity value. This paper recognises that connectivity is best defined by the interaction between species and the landscape in which they occur, and proposes a functional approach to assess connectivity. The approach utilises an incidence function model (IFM) as a spatially explicit method to assess potential species-level connectivity. The standard IFM connectivity measure is modified to account for the influence of the surrounding landscape matrix on edge impacts (through a weighted internal edge buffer) and ecological isolation (through an assessment of least-cost distance to account for landscape permeability). It has been recognised that such patch-based connectivity measures can provide misleading results when used to examine change, as they only focus on between patch movements. As a result, a modified hybrid IFM, based on a combination of patch and cell-based approaches, is developed to account for both within (intra) and between (inter) patch connectivity. The resulting probability of functional connectivity (PFC) indicator was evaluated, alongside a patch-based connectivity measure, through the application to four model landscapes based on changes (2 negative and 2 positive) to a control landscape. The four model landscapes illustrate the impact of landscape change on habitat area, edge impacts and matrix permeability. The proposed PFC indicator successfully discriminated between the two negative and the two positive changes to the control landscape, whereas, the patch-based connectivity measure detected change successfully within three of the four landscapes. The PFC indicator predicted a decrease in intra and inter-patch connectivity following habitat loss and fragmentation (negative change 1), whereas patch-based connectivity measures indicate an increase in connectivity between fragmented patches. The proposed PFC indicator offers the opportunity to take the necessary species-based perspective to examine functional connectivity, incorporating habitat preference, dispersal probability, edge impacts and ecological isolation/permeability. The urgency to assess changes in connectivity and support conservation policy means that there is little time to wait for more complete data. We believe the proposed approach provides a robust balance between the data required and the biologically meaningful indicator produced.     

Design,synthesis and evaluation of constrained tetrahydroimidazopyrimidine derivatives as antagonists of corticotropin-releasing factor type 1 receptor (CRF1R)

Several tetrahydroimidazopyrimidines were prepared using silver assisted cyclization as the key step. The binding affinities of compounds thus prepared were evaluated in vitro toward hCRF₁R. Initial lead compound 16 (K_i = 32 nM) demonstrated modest putative anxiolytic effects in the mouse canopy test. Further optimization using parallel synthesis provided compounds with K_i’s <50 nM.     

Working through polytomies: Auklets revisited

Polytomies, or phylogenetic “bushes”, are the result of a series of internodes occurring in a short period of evolutionary time (which can result in data that do not contain enough information), or data that have too much homoplasy to resolve a bifurcating branching pattern. In this study we used the Aethia auklet polytomy to explore the effectiveness of different methods for resolving polytomies: mitochondrial DNA gene choice, number of individuals per species sampled, model of molecular evolution, and AFLP loci. We recovered a fully-resolved phylogeny using NADH dehydrogenase subunit 2 (ND2) sequence data under two different Bayesian models. We were able to corroborate this tree under one model with an expanded mtDNA dataset. Effectiveness of additional intraspecific sampling varied with node, and fully 20% of the subsampled datasets failed to return a congruent phylogeny when we sampled only one or two individuals per species. We did not recover a resolved phylogeny using AFLP data. Conflict in the AFLP dataset showed that nearly all possible relationships were supported at low levels of confidence, suggesting that either AFLPs are not useful at the genetic depth of the Aethia auklet radiation (7–9% divergent in the mtDNA ND2 gene), perhaps resulting in too much homoplasy, or that the Aethia auklets have experienced incomplete lineage sorting at many nuclear loci.     

Discovery of novel sphingosine kinase-1 inhibitors. Part 2

Building on our initial work, we have identified additional novel inhibitors of sphingosine kinase-1 (SK1). These new analogs address the shortcomings found in our previously reported compounds. Inhibitors 51 and 54 demonstrated oral bioavailability in a rat PK study.