The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized,and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophilaortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophiladevelopment.

A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, ϳ3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5-non-coding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis.Cells of higher organisms can regulate gene expression at the translational level in response to a wide variety of stimuli and conditions. While much is known about the control of translational initiation and elongation in response to nutritional deprivation (1, 2) and environmental stress (2), the role of translational control in mammalian cell cycle progression is not well understood. It has long been known that specific proteins are needed at specific times during the cell cycle to ensure cell cycle progression. For the most part, the expression of these cell cycle-specific proteins is thought to be regulated at the transcriptional or posttranslational level (3).In cultured mammalian cells arrested at G 2 and M phases, the rate of total protein synthesis was markedly decreased to about 25% of the rate in non-arrested, cycling cells (4). This reduction was shown to be, at least partly, due to inhibition of the initiation step of polypeptide synthesis (4 -6). Subsequently, several eukaryotic initiation factors that regulate the assembly of 40 S ribosomes at the 5Ј ends of capped mRNAs were observed to change their phosphorylation status (7-9). Therefore, it seemed likely that inhibition of translation initiation was caused by diminishing ribosome recruitment to capped mRNAs during mitosis. Viral mRNAs whose translation is initiated by an internal ribosome entry site (IRES) 1 mechanism (10), such as polioviral and hepatitis C viral mRNAs (4,(11)(12)(13)(14) or mRNAs which lack significant structures in their 5Ј-non-coding regions (5Ј-NCRs), such as the mRNAs containing the late leader of adenovirus (15), were found to be selectively translated during mitosis due to their lessened requirement for eIF4F. More recently, additional IREScontaining mRNAs, those encoding ornithine decarboxylase and kinase p58 PITSLRE , have been reported to be selectively translated during G 2 /M of the cell cycle in cultured cells (11,12). These findings raise the question whether all IREScontaining genes are preferentially translated during mitosis and whether any of these encoded products play roles in cell cycle progression. We have begun to address these questions by genomic analysis of cellular mRNAs that are associated with mitotic polysomes and are, thus, predicted to be translated during the overall translation repression in mitosis. We determined that many, but not all, IRES elements are present in mRNAs which are selectively translated...

During apoptosis there is a substantial reduction in the rate of protein synthesis, and yet some mRNAs avoid this translational inhibition. To determine the impact that receptor-mediated cell death has on the translational efficiency of a large number of mRNAs, translational profiling was performed on MCF7 cells treated with the apoptosis-inducing ligand TRAIL. Our data indicate that approximately 3% of mRNAs remain associated with the polysomes in apoptotic cells, and genes that are involved in transcription, chromatin modification/remodeling, and the Notch signaling pathway are particularly prevalent among the mRNAs that evade translational inhibition. Internal ribosome entry segments (IRESs) were identified in several of the mRNAs that remained associated with the polysomes during apoptosis, and, importantly, these IRESs functioned efficiently in apoptotic cells. Finally, the data showed that polypyrimidine tract binding protein (PTB, a known IRES trans-acting factor or ITAF) is pivotal in regulating the apoptotic process by controlling IRES function.

Translational control during early Drosophila embryogenesis

The polysomal profiles of over 15,000 transcripts during the first ten hours after egg laying have been determined.