A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation

The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homologue of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. We report a novel function of btd and Sp1: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. We propose that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth.Dirección General de Investigación Científica y TécnicaPeer Reviewe

A functional ribonucleoprotein complex forms around the 5′ end of poliovirus RNA

The existence of a computer-predicted cloverleaf structure for the first 100 nucleotides at the 5′ end of poliovirus RNA was verified by site-directed mutagenesis and by chemical and RNAase probing. Mutations that modified the cloverleaf in the positive strand but not the negative strand were lethal to the virus. This RNA cloverleaf structure binds a cellular protein and the viral proteins 3Cᵖʳᵒ and 3Dᵖᵒⁱ. Mutations in specific regions of the RNA cloverleaf prevented this binding. Mutations in either 3Cᵖʳᵒ or the RNA that disrupted ribonucleoprotein complex formation inhibited virus growth and selectively affected positive strand RNA accumulation. Phenotypic reversion of these mutations restored the ability to form the complex. Thus, a cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production

A functional ribonucleoprotein complex forms around the 5′ end of poliovirus RNA

The existence of a computer-predicted cloverleaf structure for the first 100 nucleotides at the 5′ end of poliovirus RNA was verified by site-directed mutagenesis and by chemical and RNAase probing. Mutations that modified the cloverleaf in the positive strand but not the negative strand were lethal to the virus. This RNA cloverleaf structure binds a cellular protein and the viral proteins 3Cᵖʳᵒ and 3Dᵖᵒⁱ. Mutations in specific regions of the RNA cloverleaf prevented this binding. Mutations in either 3Cᵖʳᵒ or the RNA that disrupted ribonucleoprotein complex formation inhibited virus growth and selectively affected positive strand RNA accumulation. Phenotypic reversion of these mutations restored the ability to form the complex. Thus, a cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production

A precision oncology approach to the pharmacological targeting of mechanistic dependencies in neuroendocrine tumors

© 2018 The Author(s). We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology