SARS-CoV-2 nucleocapsid protein binds host mRNAs and attenuates stress granules to impair host stress response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is essential for viral replication, making it a promising target for antiviral drug and vaccine development. SARS-CoV-2 infected patients exhibit an uncoordinated immune response; however, the underlying mechanistic details of this imbalance remain obscure. Here, starting from a functional proteomics workflow, we cataloged the protein-protein interactions of SARS-CoV-2 proteins, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N localizes to stress granules and sequesters G3BPs away from their typical interaction partners, thus attenuating stress granule formation. We found that N binds directly to host mRNAs in cells, with a preference for 3' UTRs, and modulates target mRNA stability. We show that the N protein rewires the G3BP1 mRNA-binding profile and suppresses the physiological stress response of host cells, which may explain the imbalanced immune response observed in SARS-CoV-2 infected patients.

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.

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

CYP4V2 mutation screening in an Iranian Bietti crystalline dystrophy pedigree and evidence for clustering of CYP4V2 mutations.

PURPOSE: To report the genetic analysis of an Iranian Bietti crystalline dystrophy (BCD)-affected family, and to review previously reported mutations in the gene and assess the distribution of affected amino acids in the encoded protein. METHODS: The eleven exons of CYP4V2 were sequenced in the DNA of the proband of the Iranian BCD family. The putative disease-causing variation was screened in all affected and non-affected members. BCD causing CYP4V2 mutations previously reported in the literature were compiled, and positions of amino acids affected by nonsense and missense mutations were mapped onto the primary structure of the CYP4V2 protein. RESULTS: C.1219G > T in CYP4V2 that causes p.Glu407* was identified as cause of BCD in the Iranian family. The mutation segregated with disease status. Clinical presentations were similar among affected members, except that one patient presented with retinal macular hole. Twelve nonsense and 47 missense mutations in CYP4V2 were compiled. Inspection of distribution of amino acids affected by the mutations suggested non-random distribution and clustering of affected amino acids in nine regions of the protein, including regions that contain the heme binding site, the metal binding site, and a region between these binding sites. The most C-terminus proximal nonsense mutation affected position 482. CONCLUSIONS: This study presents results of the genetic analysis of an Iranian BCD family. Protein regions affected by mutations within the nine mutation clusters include regions well conserved among orthologous proteins and human CYP4 proteins, some of which are associated with known functions. The findings may serve to identify reasonable candidate gene region targets for gene editing therapy approaches.