Summary Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De-novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5′ splice site generally promoted exon inclusion, whereas binding near the 3′ splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.

Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with improved pharmacological properties in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity and potency, is widely used for this purpose. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide is a unique entity, and small structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.

SummaryMany biological processes involve gene-expression regulation by alternative splicing. Here, we identify the splicing factor SRSF6 as a regulator of wound healing and tissue homeostasis in skin. We show that SRSF6 is a proto-oncogene that is frequently overexpressed in human skin cancer. Overexpressing it in transgenic mice induces hyperplasia of sensitized skin and promotes aberrant alternative splicing. We identify 139 target genes of SRSF6 in skin, and show that this SR protein binds to alternative exons of the extracellular-matrix protein tenascin C pre-mRNA, promoting the expression of isoforms characteristic of invasive and metastatic cancer in a cell-type-independent manner. SRSF6 overexpression additionally results in depletion of Lgr6+ stem cells, and excessive keratinocyte proliferation and response to injury. Furthermore, the effects of SRSF6 in wound healing assayed in vitro depend on the TNC isoforms. Thus, abnormal SR-protein expression can perturb tissue homeostasis.

BackgroundDuring spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions.ResultsHere, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor’s properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions.ConclusionsThis study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0682-5) contains supplementary material, which is available to authorized users.

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central b-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including a-helix F. In order to elucidate this mechanism, we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against a-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1). One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1. When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in a-helix F and the loop connecting a-helix F and b-strand 3A as being important for the inhibitory function of PAI-1. Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.Keywords: PAI-1; vitronectin; monoclonal antibody; epitope mapping; phage-display.Plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the serine proteinases urokinase-type (uPA) and tissue-type plasminogen activator (tPA), and, as such, an important regulator of extracellular proteolysis in turnover of extracellular matrix and in fibrinolysis (reviewed in [1]). PAI-1 binds with high affinity to vitronectin (reviewed in [2,3]) and inhibits vitronectin binding of integrins [4,5] and of the uPA receptor [6,7], and is thereby able to regulate cell migration and adhesion independently of its function as a proteinase inhibitor [4,5,7±9]. The PAI-1 level in malignant tumours is one of the most informative biochemical markers of a poor prognosis (reviewed in [10,11]), and PAI-1 seems to be causally involved in tumour invasion and angiogenesis [12]. PAI-1 is therefore a potential target for anticancer therapy.PAI-1 belongs to the serpin superfamily. Serpins are composed of three b-sheets and nine a-helices, which form a scaffold supporting a solvent-exposed, about 20 amino acids long peptide loop, the reactive centre loop (RCL). Serpins and proteinases form stable complexes by interaction of the active site of the proteinases with the reactive centre peptide bond (P 1 ±P 1 H ) in the RCL (reviewed in [1,13]). There is both biochemical and structural evidence that formation of a...