Studies in Drosophila have characterized insulin receptor/phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but its function during development has remained uncertain. Here we show that inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. Hence we surmise that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions.

Members of the Myc family of proto-oncogenes have long been implicated in regulating proliferation, apoptosis and oncogenesis. Recently, transcriptional and biological studies have suggested a direct role for Myc in regulating growth. We have used dm4, a new null allele of the Drosophila diminutive (dm) gene, which encodes dMyc on the X chromosome, to investigate a role for dMyc in larval endoreplicating tissues,where cellular growth and DNA replication occur in the absence of cell division. Hemizygous dm4/Y mutants arrest as second instar larvae, and fat body nuclei of dm4/Y mutants fail to attain normal size and normal levels of DNA, resulting from a reduced frequency of S-phase. Thus, dMyc is required for endoreplication and larval growth. In support of this, dMyc, as well as its antagonist dMnt, are expressed in larval tissues in a pattern consistent with their involvement in regulating endoreplication. Overexpression of dMyc in endoreplicating cells results in dramatic increases in nuclear DNA content and cell and nucleolar size, whereas dMnt overexpression has the opposite effect. BrdU incorporation and Cyclin E protein levels continue to oscillate in dMyc-overexpressing cells, indicating that the normal cell cycle control mechanisms are not disrupted. dMyc driven growth and endoreplication are strongly attenuated when the endocycle is blocked with Cyclin E or the cdk inhibitor p21. By contrast,the ability of dMyc to promote growth and endoreplication is only partly reduced when PI3K activity is blocked, suggesting that they influence distinct growth pathways. Our results indicate that larval growth and endoreplication are coupled processes that, although linked to cell cycle control mechanisms,are regulated by dMyc and dMnt.

Mutations in stem-loop Ila of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.RNA-RNA interactions between small nuclear RNAs (snRNAs) or between snRNAs and the pre-mRNA play critical roles in the accuracy and efficiency of splicing (for reviews, see references 15 and 16). Not all of these interactions are established simultaneously, nor do they persist once established. Rather, interactions are formed, modified, disrupted, and replaced during spliceosome assembly and splicing. The dynamic relationship between U4 and U6 has been known for some time (15, 16), and more recently it has been shown that U2 also interacts with U6 (11,26,47 Structure-function studies in this region of U2 show that only one of the structures, that containing stem-loop Ila, is absolutely essential for growth, arguing that the potential to form the others must be conserved for an accessory or overspecified function (2). Direct probing of yeast U2 structure in vivo confirms that the bulk of U2 in the cell adopts the essential structure (2); however, the alternative structure can form under certain circumstances, suggesting that this region of U2 snRNA is dynamic (49).An unanticipated phenotype associated with single base changes that destabilize the essential structure is cold-sensitive growth (2). Cold sensitivity is also observed in cell splicing extracts, allowing the demonstration of a role for stem Ila in the critical step of assembly of U2 snRNPs into the spliceosome in vitro (49). Structure probing experiments show that the bulk of U2 snRNA is misfolded in the cold-sensitive mutants, so that the RNA adopts the other phylogenetically conserved structure. Surprisingly, the misfolded form predominates at both permissive and restrict...

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, we varied larval nutrition and monitored effects on cell cycle initiation and maintenance in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. We also show that ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, we demonstrate that quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition.