Increased hepatic lipid content is an early correlate of insulin resistance, and can be caused by nutrient-induced mTor activation. The latter increases basal Akt activity, leading to a self-perpetuating lipogenic cycle. We have previously shown that the developmental Notch pathway has metabolic functions in adult liver. Acute or chronic inhibition of Notch dampens hepatic glucose production and increases Akt tone, and might therefore be predicted to increase hepatic lipid content. Surprisingly, we show that constitutive liver-specific ablation of Notch signaling, or its acute inhibition with a decoy Notch1 receptor, prevents hepatosteatosis by blocking mTorc1. Conversely, Notch gain-of-function causes fatty liver through constitutive activation of mTorc1, an effect reversible by rapamycin treatment. We demonstrate that Notch signaling increases mTorc1 complex stability, augmenting mTorc1 function and Srebp1c-mediated lipogenesis. The data identify Notch as a therapeutically actionable branch point of metabolic signaling, where hepatic Akt activation can be uncoupled from steatosis.

A pro-angiogenic role for Jagged-dependent activation of Notch signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of DLL-class and JAG-class ligand/receptor interactions, and developed Notch decoys that function as ligand-specific Notch inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro and retinal angiogenesis, demonstrating JAG-dependent Notch signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG/NOTCH signaling uniquely inhibited expression of anti-angiogenic sVEFGFR-1/sFlt-1. N11-13 decoy interfered with DLL1/DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis and in tumors. Thus, blockade of JAG- or DLL-mediated Notch signaling inhibits angiogenesis by distinct mechanisms. JAG/Notch signaling positively regulates angiogenesis by suppressing sVEGFR-1/sFlt-1 and promoting mural/endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth.

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.The splicing together of exons during the maturation of mRNA from a primary transcript represents a step that is fundamental in the transfer of information from DNA to protein for most genes in higher eukaryotes. This process is catalyzed by a supramolecular particle known as the spliceosome, which serves as the site for the two transesterification reactions that remove an intron and connect two adjacent exons. The spliceosome is composed of five small nuclear RNA molecules and perhaps hundreds of proteins (20,23,33,55). Despite this complexity, much has been learned about the identity of these components, their function, and the sequence of events that culminates in exon joining (2,5,16,21). Less understood are the earliest events that initiate splicing, in which the boundaries of the intron that must be removed are identified. The absolute accuracy of this molecular recognition is critical to gene function and must depend on signals comprised of sequence and/or structural elements in the RNA substrate.In higher eukaryotes, several classes of splicing sequence elements can be distinguished based on their function and location; the splice sites themselves constitute one such class (31, 39). The 5Ј splice site is comprised of a 9-base consensus sequence that includes an almost universally conserved GU surrounded by additional nucleotides that are less well conserved. Similarly, the 3Ј splice contains a highly conserved AG surrounded by a less conserved C and G and an upstream tract of about 10 bases that is rich in pyrimidines. Splice site sequences from tens of thousands of introns have been used to define these two consensus sequences and to construct position-specific scoring matrices to evalu...