A cell-autonomous 12-hour clock coordinates nuclear speckle LLPS with proteostasis control.

Current treatments targeting individual protein quality control pathways have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common druggable target capable of global proteostasis modulation. Building upon our prior research establishing nuclear speckles as pivotal membrane-less organelles for transcriptional control of proteostasis, we aim to alleviate proteinopathies through nuclear speckle rehabilitation. We identify pyrvinium pamoate as a nuclear speckle rehabilitator that enhances protein quality control gene expression and suppresses YAP1 transcriptional activity via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate protects against retinal degeneration and tauopathy mainly by promoting autophagy and ubiquitin-proteasome activity in a SON-dependent manner without causing stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity are observed in human tauopathies. Our study provides proof-of-principle of targeting nuclear speckles to ameliorate proteinopathies.

Caloric restriction improves metabolic health but is often complicated by bone loss. We studied bone parameters in humans during a 10-day fast and identified candidate metabolic regulators of bone turnover. Pro-collagen 1 intact N-terminal pro-peptide (P1NP), a bone formation marker, decreased within 3 days of fasting. Whereas dual-energy x-ray absorptiometry measures of bone mineral density were unchanged after 10 days of fasting, high-resolution peripheral quantitative CT demonstrated remodeling of bone microarchitecture. Pathway analysis of longitudinal metabolomics data identified one-carbon metabolism as fasting dependent. In cultured osteoblasts, we tested the functional significance of one-carbon metabolites modulated by fasting, finding that methionine — which surged after 3 days of fasting — affected markers of osteoblast cell state in a concentration-dependent manner, in some instances exhibiting a U-shaped response with both low and high concentrations driving putative antibone responses. Administration of methionine to mice for 5 days recapitulated some fasting effects on bone, including a reduction in serum P1NP. In conclusion, a 10-day fast in humans led to remodeling of bone microarchitecture, potentially mediated by a surge in circulating methionine. These data support an emerging model that points to a window of optimal methionine exposure for bone health.

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

Species within the quinaria group of the genus Drosophila are models for ecological genetic studies on topics that include morphological diversity, color pattern development, and feeding behavior. Drosophila palustris and Drosophila subpalustris are closely related members within the quinaria species group that differ primarily in subtle morphological and behavioral traits. To support future evolutionary genomic studies in drosophilids, we completed new draft hybrid assemblies and annotations of the D. palustris and D. subpalustris genomes. We obtained a fragmented assembly for D. palustris (N50 = 874 Kb) and a near chromosome-scale D. subpalustris assembly (N50 = 27.5 Mbp). Assembly contiguity, repeat content, and gene annotation compared well to other published quinaria group genomes. To demonstrate the utility of our hybrid assemblies, we performed whole-genome synteny and phylogenomic comparisons within the quinaria clade. When compared to the chromosome-level Drosophila innubila assembly, the median size of syntenic blocks among quinaria group genomes was approximately 1 Mbp, although this estimate was influenced by assembly contiguity. Phylogenomic analyses estimated that the 2 major branches of the quinaria group diverged ∼17.9 MYA, with D. palustris and D. subpalustris diverging ∼1 to 2 MYA. We expect that our D. palustris and D. subpalustris assemblies will provide important additions to studies of genome evolution within Drosophilidae.