Polyadenosine RNA binding proteins (Pabs) play critical roles in regulating the polyadenylation, nuclear export, stability, and translation of cellular RNAs. Although most Pabs are ubiquitously expressed and are thought to play general roles in post-transcriptional regulation, mutations in genes encoding these factors have been linked to tissue-specific diseases including muscular dystrophy and now intellectual disability (ID). Our recent work defined this connection to ID, as we showed that mutations in the gene encoding the ubiquitously expressed Cys3His tandem zinc-finger (ZnF) Pab, ZC3H14 (Zinc finger protein, CCCH-type, number 14) are associated with non-syndromic autosomal recessive intellectual disability (NS-ARID). This study provided a first link between defects in Pab function and a brain disorder, suggesting that ZC3H14 plays a required role in regulating RNAs in nervous system cells. Here we highlight key questions raised by our study of ZC3H14 and its ortholog in the fruit fly Drosophila melanogaster, dNab2, and comment on future approaches that could provide insights into the cellular and molecular roles of this class of zinc finger-containing Pabs. We propose a summary model depicting how ZC3H14-type Pabs might play particularly important roles in neuronal RNA metabolism.

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissuespecific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.

The Drosophila Taiman (Tai) protein is homologous to the human steroid-receptor coactivators SRC1–3 and activates transcription in complex with the 20-hydroxyecdysone (20E) receptor (EcR). Tai has roles in intestinal homeostasis, germline maintenance, cell motility, and proliferation through interactions with EcR and the coactivator Yorkie (Yki). Tai also promotes invasion of tumor cells in adjacent organs, but this pro-invasive mechanism is undefined. Here, we show that Tai expression transforms sessile pupal wing cells into an invasive mass that penetrates the adjacent thorax during a period of high 20E. Candidate analysis confirms a reliance on elements of the 20E and Hippo pathways, such as Yki and the Yki-Tai target dilp8. Screening the Tai-induced wing transcriptome detects enrichment for innate immune factors, including the Spätzle (Spz) family of secreted Toll ligands that induce apoptosis during cell competition. Tai-expressing wing cells induce immune signaling and apoptosis among adjacent thoracic cells, and genetic reduction of spz, Toll, or the rpr/hid/grim pro-apoptotic factors each suppresses invasion, suggesting an intercellular Spz-Toll circuit supports killing-mediated invasion. Modeling these interactions in larval epithelia confirms that Tai kills neighboring cells via a mechanism involving Toll, Spz factors, and the Spz inhibitor Necrotic. Tai-expressing cells evade death signals by repressing the immune deficiency (IMD) pathway, which operates in parallel to Toll to control nuclear factor κB (NF-κB) activity and independently regulates JNK activity. In sum, these findings suggest that Tai promotes competitive cell killing via Spz-Toll and that this killing mechanism supports pathologic intertissue invasion in Drosophila.

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a "bind and discard" mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.

The Drosophila domino locus encodes DNA-dependent ATPases of the SWI2/SNF2 class. This class of chromatin remodeler is associated with an array of cellular activities encompassing transcription, replication, repair and recombination. Moreover, domino was observed initially to maintain a repressive chromatin state via genetic interaction studies with homeotic genes. Although domino mutations were also characterized with a cell death phenotype, its association with a death pathway has not been investigated. Here we have used targeted RNA interference to depress domino function in the wing. Resultant wing damage phenotypes were found to be enhanced through overexpression of pro-apoptotic loci, and suppressed through loss of function of these loci. Loss of wing margin and blade tissue was correlated with activation of the effector Caspase Dcp-1, a marker for apoptosis. The affected wing regions also exhibited lower levels of the DIAP1 protein, an inhibitor of apoptosis. The lower level of DIAP1 protein was not correlated with an effect on the activity of a DIAP1 gene transgenic reporter (thread-LacZ), suggesting that loss of DIAP1 occurred post transcriptionally. In some cases excessive cell proliferation within the targeted tissue, measured through BrdU incorporation, was also observed. Finally, we used a transgenic reporter construct to monitor the chromatin state upstream of the proapoptotic reaper locus. In genotypes exhibiting targeted domino loss and wing phenotypes, we observed increased reporter activity only in the affected areas. These data support the conclusion that domino normally functions to maintain pro-apoptotic genes in a repressed state.

A ten-eleven translocation (TET) ortholog exists as a DNA N 6 -methyladenine (6mA) demethylase (DMAD) in Drosophila. However, the molecular roles of 6mA and DMAD remain unexplored. Through genome-wide 6mA and transcriptome profiling in Drosophila brains and neuronal cells, we found that 6mA may epigenetically regulate a group of genes involved in neurodevelopment and neuronal functions. Mechanistically, DMAD interacts with the Trithorax-related complex protein Wds to maintain active transcription by dynamically demethylating intragenic 6mA. Accumulation of 6mA by depleting DMAD coordinates with Polycomb proteins and contributes to transcriptional repression of these genes. Our findings suggest that active 6mA demethylation by DMAD plays essential roles in fly CNS by orchestrating through added epigenetic mechanisms. A DNA N 6 -methyladenine (6mA) demethylase (DMAD) was found in Drosophila to actively remove 6mA. Yao et al. demonstrate that DMAD depletion in neurons leads to impaired neurodevelopment accompanied by 6mA accumulation. DMAD and 6mA epigenetically modulate a group of neuronal genes by coordinating with the Trithorax and Polycomb histone modifiers.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNAbinding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.

Cancer stem cells exert enormous influence on neoplastic behavior, in part by governing asymmetric cell division and the balance between self-renewal and multipotent differentiation. Growth is favored by deregulated stem cell division, which enhances the self-renewing population and diminishes the differentiation program. Mutation of a single gene in Drosophila, Brain Tumor (Brat), leads to disrupted asymmetric cell division resulting in dramatic neoplastic proliferation of neuroblasts and massive larval brain overgrowth. To uncover the mechanisms relevant to deregulated cell division in human glioma stem cells, we first developed a novel adult Drosophila brain tumor model using brat-RNAi driven by the neuroblast-specific promoter inscuteable. Suppressing Brat in this population led to the accumulation of actively proliferating neuroblasts and a lethal brain tumor phenotype. brat-RNAi caused upregulation of Notch signaling, a node critical for self-renewal, by increasing protein expression and enhancing nuclear transport of Notch intracellular domain (NICD). In human glioblastoma, we demonstrated that the human ortholog of Drosophila Brat, tripartite motif-containing protein 3 (TRIM3), similarly suppressed NOTCH1 signaling and markedly attenuated the stem cell component. We also found that TRIM3 suppressed nuclear transport of active NOTCH1 (NICD) in glioblastoma and demonstrated that these effects are mediated by direct binding of TRIM3 to the Importin complex. Together, our results support a novel role for Brat/TRIM3 in maintaining stem cell equilibrium and suppressing tumor growth by regulating NICD nuclear transport.

Background The Drosophila archipelago gene (ago) encodes the specificity component of a ubiquitin-ligase that targets the Cyclin E and dMyc proteins for degradation. Its human ortholog Fbw7 is commonly lost in many cancers, suggesting that failure to degrade ago/Fbw7 targets leads to excess tissue growth. Results Here we show that although loss of ago induces hyperplasia of some organs, it paradoxically shrinks the size of the adult eye. We find that this reflects a requirement for ago to restrict apoptotic activity of the rbf1/e2f1 pathway adjacent to the eye-specific morphogenetic furrow: ago mutant cells display elevated de2f1 activity, express the pro-death dE2f1 targets hid and rpr, and undergo high rates of apoptosis. This death and the resulting small-eye phenotype are dependent on rbf1, de2f1, hid, and the rbf1/de2f1 regulators cyclin E and dacapo, but are independent of dp53. A transactivation-deficient de2f1 allele blocks MF-associated apoptosis of ago mutant cells but does not retard their clonal overgrowth, indicating that intact de2f1 function is required for the death but not overproliferation of ago cells. Alleles of EGFR and wg pathway components further modulate the ago apoptotic and eye size phenotypes, suggesting these pathways control rates of de2f1-driven apoptosis among ago mutant cells. Conclusions These data show that ago loss requires a collaborating block in cell death to efficiently drive tissue overgrowth and that this conditional growth-suppressor phenotype reflects a role for the gene in restricting apoptotic output of the rbf1/de2f1 pathway. Moreover, the susceptibility of ago mutant cells to succumb to this apoptotic program appears to depend on local variations in extracellular signaling that could thus determine tissue-specific fates of ago mutant cells.