Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

Discrete and Continuous Cell Identities of the Adult Murine Striatum

The striatum is a large brain region containing two major cell types: D1 (dopamine receptor 1) and D2 (dopamine receptor 2) expressing spiny projection neurons (SPNs). We generated a cell type atlas of the adult murine striatum using single-cell RNA-seq of SPNs combined with quantitative RNAin situhybridization (ISH). We developed a novel computational pipeline that distinguishes discrete versus continuous cell identities in scRNA-seq data, and used it to show that SPNs in the striatum can be classified into four discrete types that reside in discrete anatomical clusters or are spatially intermingled. Within each discrete type, we find multiple independent axes of continuous cell identity that map to spatial gradients and whose genes are conserved between discrete types. These gradients correlate well to previously-mapped gradients of connectivity. Using these insights, we discovered multiple novel spatially localized region of the striatum, one of which contains patch-D2 SPNs that expressTac1, Htr7, andTh. Intriguingly, we found one subtype that strongly co-expresses both D1 and D2 dopamine receptors, and uniquely expresses a rare D2 receptor splice variant. These results collectively suggest an organizational principal of neuron identity in which major neuron types can be separated into discrete classes with little overlap and no implied spatial relationship. However these discrete classes are then continuously subdivided by multiple spatial gradients of expression defining anatomical location via a combinatorial mechanism. Finally, they suggest that neuronal circuitry has a substructure at far higher resolution than is typically interrogated which is defined by the precise identity and location of a neuron.

Transcription activator-coactivator specificity is mediated by a large and dynamic fuzzy protein-protein complex

SUMMARYTranscription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient activation by yeast Gcn4 requires tandem Gcn4 ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous, containing nearly all possible AD-ABD interactions. This complex forms using a dynamic fuzzy protein-protein interface where ADs use hydrophobic residues to bind hydrophobic surfaces of the ABDs in multiple orientations. This combinatorial mechanism allows individual interactions of low affinity and specificity to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators and allows great flexibility in combinations of activators that synergize to regulate genes with variable coactivator requirements.

Causing something to be one way rather than another

Purpose – The purpose of this paper is to suggest a definition of genetic information by taking into account the debate surrounding it. Particularly, the objections raised by Developmental Systems Theory (Griffiths, 2001; Oyama 1985; Griffiths and Knight 1998) to Teleosemantic endorsements of the notion of genetic information (Sterelny et al. 1996; Maynard Smith, 2000; Jablonka, 2002) as well as deflationist approaches which suggest to ascribe the notion of genetic information a heuristic value at most, and to reduce it to that of causality (Godfrey-Smith, 2000; Boniolo, 2003, 2008). Design/methodology/approach – The paper presents the notion of genetic information through its historical evolution and analyses it with the conceptual tools offered by philosophical theories of causation on one side (“causation as influence,” Woodward, 2010; Waters, 2007; Lewis, 2000) and linguistics on the other (“double articulation” Martinet, 1960). Findings – The concept of genetic information is defined as a special kind of cause which causes something to be one way rather than another, by combining elementary units one way rather than another. Tested against the notion of “genetic error” this definition demonstrates to provide an exhaustive account of the common denominators associated with the notion of genetic information: causal specificity; combinatorial mechanism; arbitrariness. Originality/value – The definition clarifies how the notion of information is understood when applied to genetic phenomena and also contributes to the debate on the notion of information, broadly meant, which is still affected by lack of consensus (Floridi, 2013).

Low oxygen levels induce the expression of the embryonic morphogen Nodal

Low oxygen (O2) levels characterize the microenvironment of both stem cells and rapidly growing tumors. Moreover, hypoxia is associated with the maintenance of stem cell–like phenotypes and increased invasion, angiogenesis and metastasis in cancer patients. Metastatic cancers, such as breast cancer and melanoma, aberrantly express the embryonic morphogen Nodal, and the presence of this protein is correlated with metastatic disease. In this paper, we demonstrate that hypoxia induces Nodal expression in melanoma and breast cancer cells concomitant with increased cellular invasion and angiogenic phenotypes. Of note, Nodal expression remains up-regulated up to 48 h following reoxygenation. The oxygen-mediated regulation of Nodal expression occurs via a combinatorial mechanism. Within the first 24 h of exposure to low O2, there is an increase in protein stability. This increase in stability is accompanied by an induction of transcription, mediated by the HIF-1α–dependent activation of Notch-responsive elements in the node-specific enhancer of the Nodal gene locus. Finally, Nodal expression is maintained upon reoxygenation by a canonical SMAD-dependent feed-forward mechanism. This work provides insight into the O2-mediated regulation of Nodal, a key stem cell–associated factor, and reveals that Nodal may be a target for the treatment and prevention of hypoxia-induced tumor progression.