Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.[Keywords: Alternative splicing; neuronal development; nonsense-mediated decay; polypyrimidine tract-binding proteins; splicing microarray; ultraconserved element] Supplemental material is available at http://www.genesdev.org. Alternative pre-mRNA splicing is a common mechanism for diversifying genetic output in metazoan organisms (Black 2003;Matlin et al. 2005). Alternative choices in exons and splice sites can create substantial changes in the encoded protein and its activity. Changes in splicing can also affect downstream regulatory processes such as nonsense-mediated decay (NMD), and thus direct additional levels of post-transcriptional gene regulation (Lewis et al. 2003;Lejeune and Maquat 2005;Hughes 2006). Transcripts exhibiting multiple splicing patterns are especially prevalent in the mammalian nervous system, where alternative splicing affects important processes such as axon guidance, synaptogenesis, and the regulation of membrane physiology (Black and Grabowski 2003;Lipscombe 2005;. The choice of splicing pattern within a transcript is generally controlled by RNA-binding proteins that bind to the pre-mRNA to enhance or silence particular splicing events (Black 2003;Matlin et al. 2005). Some splicing regulators are expressed in a tissue-specific manner and have been sh...

Postsynaptic density protein 95 (PSD-95) is essential for synaptic maturation and plasticity. Although its synaptic regulation is widely studied, the control of PSD-95 cellular expression is not understood. We find that Psd-95 is controlled post-transcriptionally during neural development. Psd-95 is transcribed early in mouse embryonic brain, but most of its product transcripts are degraded. The polypyrimidine tract binding proteins, PTBP1 and PTBP2, repress Psd-95 exon 18 splicing, leading to premature translation termination and nonsense-mediated mRNA decay (NMD). The loss first of PTBP1 and then of PTBP2 during embryonic development allows splicing of Exon 18 and expression of PSD-95 late in neuronal maturation. Re-expression of PTBP1 or PTBP2 in differentiated neurons inhibits PSD-95 expression and impairs development of glutamatergic synapses. Thus, expression of PSD-95 during early neural development is controlled at the RNA level by two PTB proteins whose sequential down-regulation is necessary for synapse maturation.

[Keywords: Locked nucleic acids; alternative splicing; miR-133; microRNAs; myogenesis; nPTB] Supplemental material is available at http://www.genesdev.org.

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.Alternative splicing allows multiple functionally distinct mRNAs and proteins to be generated from a single gene, greatly enhancing the coding potential of the genome. Splicing patterns are controlled by RNA binding proteins that recognize specific splicing enhancer and silencer elements in the premRNA to alter spliceosome assembly, and variation in the expression of these proteins leads to tissue-specific exon use (5, 38). Splicing within a single cell can also be dynamically controlled by extracellular stimuli, although how this information is transmitted to splicing regulatory proteins is not yet known (55).Sam68 is a nuclear RNA binding protein implicated in various aspects of mRNA metabolism, including splicing, nuclear export, somatodendritic transport, and translation. Sam68 belongs to the family of GSG (GRP33, Sam68, GLD1) or STAR (signal transduction and activation of RNA) domain proteins. This domain includes a central KH (hnRNP K homology) RNA binding domain flanked by conserved N and C termini (36,54,59). The GSG domain in Sam68 binds to RNA motifs that are rich in A or U, such as UAAA or UUUA, and also mediates homodimerization (9, 31). Sam68 contains a variety of other protein domains that allow its interaction and modification by multiple signaling pathways. These many regulatory interactions and posttranslational modifications affect the RNA binding activity and localization of Sam68 and make it an appealing molecule for transducing information from signaling systems to pathways of mRNA metabolism (13,33,46,51,60).Sam68 is localized primarily in the nucleus as observed by immunofluorescence, consistent with its role in alternative splicing. Sam68 helps regulate the splicing of CD44 variable exon v5 in response to phosphorylation by extracellular signalregulated kinase in T lymphoma cells (39). Sam68 binds to exonic regulatory elements of v5 and cooperates wi...

Macrophage-mediated phagocytosis and cytokine production represent the front lines of resistance to bacterial invaders. A key feature of this pro-inflammatory response in mammals is the complex remodeling of cellular metabolism towards aerobic glycolysis. Although the function of bactericidal macrophages is highly conserved, the metabolic remodeling of insect macrophages remains poorly understood. Here, we used adults of the fruit fly Drosophila melanogaster to investigate the metabolic changes that occur in macrophages during the acute and resolution phases of Streptococcus-induced sepsis. Our studies revealed that orthologs of Hypoxia inducible factor 1α (HIF1α) and Lactate dehydrogenase (LDH) are required for macrophage activation, their bactericidal function, and resistance to infection, thus documenting the conservation of this cellular response between insects and mammals. Further, we show that macrophages employing aerobic glycolysis induce changes in systemic metabolism that are necessary to meet the biosynthetic and energetic demands of their function and resistance to bacterial infection.

L-2-hydroxyglutarate (L-2HG) has emerged as a putative oncometabolite that is capable of inhibiting enzymes involved in metabolism, chromatin modification, and cell differentiation. However, despite the ability of L-2HG to interfere with a broad range of cellular processes, this molecule is often characterized as a metabolic waste product. Here, we demonstrate that Drosophila larvae use the metabolic conditions established by aerobic glycolysis to both synthesize and accumulate high concentrations of L-2HG during normal developmental growth. A majority of the larval L-2HG pool is derived from glucose and dependent on the Drosophila estrogen-related receptor (dERR), which promotes L-2HG synthesis by up-regulating expression of the Drosophila homolog of lactate dehydrogenase (dLdh). We also show that dLDH is both necessary and sufficient for directly synthesizing L-2HG and the Drosophila homolog of L-2-hydroxyglutarate dehydrogenase (dL2HGDH), which encodes the enzyme that breaks down L-2HG, is required for stage-specific degradation of the L-2HG pool. In addition, dLDH also indirectly promotes L-2HG accumulation via synthesis of lactate, which activates a metabolic feedforward mechanism that inhibits dL2HGDH activity and stabilizes L-2HG levels. Finally, we use a genetic approach to demonstrate that dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, our studies demonstrate that growing animal tissues synthesize L-2HG in a controlled manner, reveal a mechanism that coordinates glucose catabolism with L-2HG synthesis, and establish the fly as a unique model system for studying the endogenous functions of L-2HG during cell growth and proliferation.ne of the hallmarks of cancer is a dramatic reprograming of cellular metabolism that results in enhanced biosynthesis (1). These metabolic changes are particularly apparent in tumors that use the Warburg effect, also referred to as aerobic glycolysis, a metabolic program characterized by elevated levels of glucose consumption and enhanced lactate production (1, 2). By activating aerobic glycolysis, tumors are able to synthesize macromolecules rapidly from glycolytic intermediates. In addition, elevated levels of lactate dehydrogenase (LDH) activity allow proliferating cells to synthesize lactate and maintain the NAD + levels required for high rates of glucose catabolism and biomass production (1).The metabolic reprogramming of cancer cells, however, extends beyond biosynthesis, as many tumors also generate progrowth metabolites, or oncometabolites, that promote tumor formation via nonmetabolic means. Most notable among these compounds is D-2-hydroxyglutarate (D-2HG), which is associated with cancers such as gliomas and acute myelogenous leukemias (3). Although D-2HG is generated as a normal byproduct of γ-hydroxybutyrate metabolism (4), oncogenic D-2HG production is the result of neomorphic mutations in the active site of isocitrate dehydrogenas...

Steroid hormones and their nuclear receptors drive developmental transitions in diverse organisms, including mammals. In this study, we show that the Drosophila steroid hormone 20-hydroxyecdysone (20E) and its nuclear receptor directly activate transcription of the evolutionarily conserved let-7-complex (let-7-C) locus, which encodes the co-transcribed microRNAs miR-100, let-7 and miR-125. These small RNAs post-transcriptionally regulate the expression of target genes, and are required for the remodeling of the Drosophila neuromusculature during the larval-to-adult transition. Deletion of three 20E responsive elements located in the let-7-C locus results in reduced levels of let-7-C microRNAs, leading to neuromuscular and behavioral defects in adults. Given the evolutionary conservation of let-7-C microRNA sequences and temporal expression profiles, these findings indicate that steroid hormone-coupled control of let-7-C microRNAs is part of an ancestral pathway controlling the transition from larval-to-reproductive animal forms.

Messenger RNAs (mRNAs) often contain binding sites for multiple, different microRNAs (miRNAs). However, the biological significance of this feature is unclear, since such co-targeting miRNAs could function coordinately, independently, or redundantly with one another. Here, we show that two co-transcribed Drosophila miRNAs, let-7 and miR-125, non-redundantly regulate a common target, the transcription factor Chronologically Inappropriate Morphogenesis (Chinmo). We first characterize novel adult phenotypes associated with loss of both let-7 and miR-125, which are derived from a common, polycistronic transcript that also encodes a third miRNA, miR-100. Consistent with the coordinate upregulation of all three miRNAs in aging flies, these phenotypes include brain degeneration and shortened lifespan. However, transgenic rescue analysis reveal separable roles for these miRNAs: adult miR-125 but not let-7 mutant phenotypes are associated with ectopic Chinmo expression in adult brains and are suppressed by chinmo reduction. In contrast, let-7 is predominantly responsible for regulating chinmo during nervous system formation. These results indicate that let-7 and miR-125 function during two distinct stages, development and adulthood, rather than acting at the same time. These different activities are facilitated by an increased rate of processing of let-7 during development and a lower rate of decay of the accumulated miR-125 in the adult nervous system. Thus, this work not only establishes a key role for the highly conserved miR-125 in aging. It also demonstrates that two co-transcribed miRNAs function independently during distinct stages to regulate a common target, raising the possibility that such biphasic control may be a general feature of clustered miRNAs.