The Notch receptor mediates a short-range signal that regulates many cell fate decisions. The misregulation of Notch has been linked to cancer and to developmental disorders. Upon binding to its ligands, Delta (Dl) or Serrate (Ser), the Notch ectodomain is shed by the action of an ADAM protease. The Notch intracellular domain is subsequently released proteolytically from the membrane by Presenilin and translocates to the nucleus to activate the transcription factor, Suppressor of Hairless. We show in Drosophila that Notch signaling is limited by the activity of two Nedd4 family HECT domain proteins, Suppressor of deltex [Su(dx)] and DNedd4. We rule out models by which Su(dx) downregulates Notch through modulating Deltex or by limiting the adherens junction accumulation of Notch. Instead, we show that Su(dx) regulates the postendocytic sorting of Notch within the early endosome to an Hrs- and ubiquitin-enriched subdomain en route to the late endosome. We propose a model in which endocytic sorting of Notch mediates a decision between its activation and downregulation. Such intersections between trafficking routes may provide key points at which other signals can modulate Notch activity in both normal development and in the pathological misactivation of Notch.

Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Here, we found that Dx was required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx required N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activated a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that was independent of the Delta (Dl)/Serrate (Ser) ligands- or Su(H). Dx caused N to be moved from the apical cell surface into the late-endosome, where it accumulated stably and co-localized with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibited the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)-independent N signaling.

Notch is a vitally important signalling receptor controlling cell fate determination and pattern formation in numerous ways during development of both invertebrate and vertebrate species. An intriguing pathway for the Notch signal has emerged where, after ligand-dependent proteolysis, an intracellular fragment of the receptor itself translocates to the nucleus to regulate gene expression. The nuclear activity of the Notch intracellular domain is linked to complexes regulating chromatin organization through histone deacetylation and acetylation. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. Regulation occurs at multiple levels including patterns of ligand and receptor expression, Notch-ligand interactions, trafficking of the receptor and ligands, and covalent modifications including glycosylation, phosphorylation and ubiquitination. Several Notch regulatory proteins have conserved domains that link them to the ubiquitination pathway, and ubiquitination of the Notch intracellular domain has recently been linked to its degradation. Different proteolytically derived isoforms of Notch have also been identified that may be involved in alternative Notch-dependent signals or regulatory mechanisms, and differences between the four mammalian Notch homologues are beginning to be appreciated.

Specific recognition of splice sites within metazoan mRNA precursors (pre-mRNAs) is a potential stage for gene regulation by alternative splicing. Splicing factors of the SR protein family play a major role in this regulation, as they are required for early recognition of splice sites during spliceosome assembly. Here, we describe the characterization of RSF1, a splicing repressor isolated from Drosophila, that functionally antagonizes SR proteins. Like the latter, RSF1 comprises an amino-terminal RRM-type RNA-binding domain, whereas its carboxy-terminal part is enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain). RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation. RSF1 interacts, through its GRS domain, with the RS domain of the SR protein SF2/ASF and prevents the latter from cooperating with the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in binding pre-mRNA. Furthermore, overproduction of RSF 1 in the fly rescues several developmental defects caused by overexpression of the splicing activator SR protein B52/ SRp55. Therefore, RSF1 may correspond to the prototypical member of a novel family of general splicing repressors that selectively antagonize the effect of SR proteins on 5 splice-site recognition.[Key Words: pre-mRNA splicing; RNA-binding proteins; SF2/ASF; U1 snRNP; RSF1; B52/SRp55] Received September 25, 1998; revised version accepted January 28, 1999.Pre-mRNA splicing is an essential step in the expression of most metazoan protein-coding genes, often regulated in a cell-type specific or developmental manner. The precision and efficiency of the splicing reaction result from a dynamic series of interactions among the U1, U2, U4/ U6, and U5 small nuclear ribonucleoprotein (snRNP) particles, non-snRNPs, and the pre-mRNA that lead to the formation of the spliceosome, the ribonucleoprotein structure in which the accurate excision of intervening sequences (introns) takes place (Sharp 1994; Krämer 1996;Staley and Guthrie 1998). Although most of the biochemical events involved in constitutive pre-mRNA splicing are being elucidated, the complex regulation underlying the selection of alternative exons is less well understood (Adams et al. 1996;Wang and Manley 1997). Both structural features and sequence content of premRNAs appear to be involved (Green 1991). In the past few years, a class of purine-rich exonic elements known as exonic splicing enhancers (ESEs) has been identified in a number of pre-mRNAs (Katz and Skalka 1990;Lavigueur et al. 1993; Sun et al. 1993a,b;Watakabe et al. 1993;Xu et al. 1993;Caputi et al. 1994;Dirksen et al. 1994Dirksen et al. , 1995Tanaka et al. 1994;Ramchatesingh et al. 1995;Staffa and Cochrane 1995;Staffa et al. 1997). These sequences are likely to play a critical role in the initial recognition and pairing of the 5Ј and 3Ј splice sites (Tian and Maniatis 1993;Staknis and Reed 1994;Berget 1995). They are expected to bind a set of RNA-bin...

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.