Ecd promotes U5 snRNP maturation and Prp8 stability.

Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes.

Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing.

The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

A Drosophila model to study retinitis pigmentosa pathology associated with mutations in the core splicing factor Prp8.

Retinitis pigmentosa (RP) represents genetically heterogeneous and clinically variable disease characterized by progressive degeneration of photoreceptors resulting in a gradual loss of vision. The autosomal dominant RP type 13 (RP13) has been linked to the malfunction of PRPF8, an essential component of the spliceosome. Over 20 different RP-associated PRPF8 mutations have been identified in human patients. However, the cellular and molecular consequences of their expression in vivo in specific tissue contexts remain largely unknown. Here, we establish a Drosophila melanogaster model for RP13 by introducing the nine distinct RP mutations into the fly PRPF8 ortholog prp8 and express the mutant proteins in precise spatiotemporal patterns using the Gal4/UAS system. We show that all nine RP-Prp8 mutant proteins negatively impact developmental timing, albeit to a different extent, when expressed in the endocrine cells producing the primary insect moulting hormone. In the developing eye primordium, uncommitted epithelial precursors rather than differentiated photoreceptors appeared sensitive to Prp8 malfunction. Expression of the two most pathogenic variants, Prp8S>F and Prp8H>R, induced apoptosis causing alterations to the adult eye morphology. The affected tissue mounted stress and cytoprotective responses, while genetic programs underlying neuronal function were attenuated. Importantly, the penetrance and expressivity increased under prp8 heterozygosity. In contrast, blocking apoptosis alleviated cell loss but not the redox imbalance. Remarkably, the pathogenicity of the RP-Prp8 mutations in Drosophila correlates with the severity of clinical phenotypes in patients carrying the equivalent mutations, highlighting the suitability of the Drosophila model for in-depth functional studies of the mechanisms underlying RP13 etiology.This article has an associated First Person interview with the first author of the paper.

Identification of aspirin analogues that repress NF-κB signalling and demonstrate anti-proliferative activity towards colorectal cancer in vitro and in vivo.

Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.