No abstract

Knowledge of how the brain achieves its diverse central control of basic physiology is severely limited by the virtual absence of appropriate cell models. Isolation of clonal populations of unique peptidergic neurons from the hypothalamus will facilitate these studies. Herein we describe the mass immortalization of mouse primary hypothalamic cells in monolayer culture, resulting in the generation of a vast representation of hypothalamic cell types. Subcloning of the heterogeneous cell populations resulted in the establishment of 38 representative clonal neuronal cell lines, of which 16 have been further characterized by analysis of 28 neuroendocrine markers. These cell lines represent the first available models to study the regulation of neuropeptides associated with the control of feeding behavior, including neuropeptide Y, ghrelin, urocortin, proopiomelanocortin, melanin-concentrating hormone, neurotensin, proglucagon, and GHRH. Importantly, a representative cell line responds appropriately to leptin stimulation and results in the repression of neuropeptide Y gene expression. These cell models can be used for detailed molecular analysis of neuropeptide gene regulation and signal transduction events involved in the direct hormonal control of unique hypothalamic neurons, not yet possible in the whole brain. Such studies may contribute information necessary for the strategic design of therapeutic interventions for complex neuroendocrine disorders, such as obesity.

Examples of associations between human disease and defects in pre–messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies.

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x S splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.