Evaluating bias-reducing protocols for RNA sequencing library preparation.

BACKGROUND: Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol).A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure. RESULTS: A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias. CONCLUSIONS: The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways.

Therapeutic improvement of scarring: mechanisms of scarless and scar-forming healing and approaches to the discovery of new treatments.

Scarring in the skin after trauma, surgery, burn or sports injury is a major medical problem, often resulting in loss of function, restriction of tissue movement and adverse psychological effects. Whilst various studies have utilised a range of model systems that have increased our understanding of the pathways and processes underlying scar formation, they have typically not translated to the development of effective therapeutic approaches for scar management. Existing treatments are unreliable and unpredictable and there are no prescription drugs for the prevention or treatment of dermal scarring. As a consequence, scar improvement still remains an area of clear medical need. Here we describe the basic science of scar-free and scar-forming healing, the utility of pre-clinical model systems, their translation to humans, and our pioneering approach to the discovery and development of therapeutic approaches for the prophylactic improvement of scarring in man.

An extended set of PRDM1/BLIMP1 target genes links binding motif type to dynamic repression.

The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP1) regulates gene expression and cell fate. The DNA motif bound by BLIMP1 in vitro overlaps with that of interferon regulatory factors (IRFs), which respond to inflammatory/immune signals. At such sites, BLIMP1 and IRFs can antagonistically regulate promoter activity. In vitro motif selection predicts that only a subset of BLIMP1 or IRF sites is subject to antagonistic regulation, but the extent to which antagonism occurs is unknown, since an unbiased assessment of BLIMP1 occupancy in vivo is lacking. To address this, we identified an extended set of promoters occupied by BLIMP1. Motif discovery and enrichment analysis demonstrate that multiple motif variants are required to capture BLIMP1 binding specificity. These are differentially associated with CpG content, leading to the observation that BLIMP1 DNA-binding is methylation sensitive. In occupied promoters, only a subset of BLIMP1 motifs overlap with IRF motifs. Conversely, a distinct subset of IRF motifs is not enriched amongst occupied promoters. Genes linked to occupied promoters containing overlapping BLIMP1/IRF motifs (e.g. AIM2, SP110, BTN3A3) are shown to constitute a dynamic target set which is preferentially activated by BLIMP1 knock-down. These data confirm and extend the competitive model of BLIMP1 and IRF interaction.

Predicting druggable binding sites at the protein-protein interface.

Protein-protein interfaces are highly attractive targets for drug discovery because they are involved in a large number of disease pathways where therapeutic intervention would bring widespread benefit. Recent successes have challenged the widely held belief that these targets are 'undruggable'. The pocket finding algorithms described here show marked differences between the binding pockets that define protein-protein interactions (PPIs) and those that define protein-ligand interactions (PLIs) of currently marketed drugs. In the case of PPIs, drug discovery methods that simultaneously target several small pockets at the protein-protein interface are likely to increase the chances of success in this new and important field of therapeutics.

Predicting protein interaction sites: binding hot-spots in protein-protein and protein-ligand interfaces.

MOTIVATION: Protein assemblies are currently poorly represented in structural databases and their structural elucidation is a key goal in biology. Here we analyse clefts in protein surfaces, likely to correspond to binding 'hot-spots', and rank them according to sequence conservation and simple measures of physical properties including hydrophobicity, desolvation, electrostatic and van der Waals potentials, to predict which are involved in binding in the native complex. RESULTS: The resulting differences between predicting binding-sites at protein-protein and protein-ligand interfaces are striking. There is a high level of prediction accuracy (< or =93%) for protein-ligand interactions, based on the following attributes: van der Waals potential, electrostatic potential, desolvation and surface conservation. Generally, the prediction accuracy for protein-protein interactions is lower, with the exception of enzymes. Our results show that the ease of cleft desolvation is strongly predictive of interfaces and strongly maintained across all classes of protein-binding interface.

Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling.

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.