High-throughput sensitive screening of small molecule modulators of microexon alternative splicing using dual Nano and Firefly luciferase reporters

Abstract Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system. [Display omitted] •SARS-CoV-2 infection in induced lung cells is characterized by phosphoproteomics•Analysis of response reveals host cell signaling and protein expression profile•Comparison to studies in undifferentiated cell lines shows unique pathology in iAT2s•Systems-level predictions find druggable pathways that can impede viral life cycle Hekman et al. describe how a layer of primary stem cells (iAT2s) recapitulating lung biology responds to infection with SARS-CoV-2. They compare their work to previous studies with immortalized cell lines. Their data predict what effect the virus has on a lung cell and which drugs may slow infection