Xrp1 governs the stress response program to spliceosome dysfunction.

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP-deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction and mediator of the stress-induced cellular senescence program.

The removal of introns and the joining of exons into mature mRNA by the spliceosome is crucial in regulating gene expression, simultaneously safeguarding genome integrity and enhancing proteome diversity in multicellular organisms. Spliceosome dysfunction is thus associated with various diseases and organismal aging. Our study describes the cascade of events in response to spliceosome dysfunction. We identified two transcription factors as drivers of a stress response program triggered by spliceosome dysfunction, which dramatically remodel gene expression to protect tissue integrity and induce a senescent-like state in damaged cells prior to their inevitable elimination. Together, we highlight the indispensable role of spliceosomes in maintaining homeostasis and implicate spliceosome dysfunction in senescent cell accumulation associated with the pathomechanisms of spliceopathies and aging.

Shared enhancer gene regulatory networks between wound and oncogenic programs.

Wound response programs are often activated during neoplastic growth in tumors. In both wound repair and tumor growth, cells respond to acute stress and balance the activation of multiple programs, including apoptosis, proliferation, and cell migration. Central to those responses are the activation of the JNK/MAPK and JAK/STAT signaling pathways. Yet, to what extent these signaling cascades interact at the cis-regulatory level and how they orchestrate different regulatory and phenotypic responses is still unclear. Here, we aim to characterize the regulatory states that emerge and cooperate in the wound response, using the Drosophila melanogaster wing disc as a model system, and compare these with cancer cell states induced by rasV12scrib-/- in the eye disc. We used single-cell multiome profiling to derive enhancer gene regulatory networks (eGRNs) by integrating chromatin accessibility and gene expression signals. We identify a 'proliferative' eGRN, active in the majority of wounded cells and controlled by AP-1 and STAT. In a smaller, but distinct population of wound cells, a 'senescent' eGRN is activated and driven by C/EBP-like transcription factors (Irbp18, Xrp1, Slow border, and Vrille) and Scalloped. These two eGRN signatures are found to be active in tumor cells at both gene expression and chromatin accessibility levels. Our single-cell multiome and eGRNs resource offers an in-depth characterization of the senescence markers, together with a new perspective on the shared gene regulatory programs acting during wound response and oncogenesis.

Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes.

Post-transcriptional gene silencing using double-stranded RNA has revolutionized the field of functional genetics, allowing fast and easy disruption of gene function in various organisms. In Drosophila, many transgenic RNAi lines have been generated in large-scale efforts, including the Drosophila Transgenic RNAi Project (TRiP), to facilitate in vivo knockdown of virtually any Drosophila gene with spatial and temporal resolution. The available transgenic RNAi lines represent a fundamental resource for the fly community, providing an unprecedented opportunity to address a vast range of biological questions relevant to basic and biomedical research fields. However, caution should be applied regarding the efficiency and specificity of the RNAi approach. Here, we demonstrate that pVALIUM10-based RNAi lines, representing ∼13% of the total TRiP collection (1,808 of 13,410 pVALIUM TRiP-based RNAi lines), cause unintended off-target silencing of transgenes expressed from Gateway destination vectors. The silencing is mediated by targeting attB1 and attB2 sequences generated via site-specific recombination and included in the transcribed mRNA. Deleting these attB sites from the Gateway expression vector prevents silencing and restores expected transgene expression.

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.

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.