G3BP isoforms differentially affect stress granule assembly and gene expression during cellular stress.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these membraneless organelles is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs form SGs following stress-induced translational arrest.

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing , we generated full and partial IGHMBP2 deletion K562 cell lines.

Protein-protein interactions with G3BPs drive stress granule condensation and gene expression changes under cellular stress.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest.

A ubiquitous GC content signature underlies multimodal mRNA regulation by DDX3X.

The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs.

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a non-essential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines.

A ubiquitous GC content signature underlies multimodal mRNA regulation by DDX3X.

The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs.

Mapping the genetic landscape of DNA double-strand break repair.

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes.

Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning.

Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles.

Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes.

Exploring genetic interaction manifolds constructed from rich single-cell phenotypes.

How cellular and organismal complexity emerges from combinatorial expression of genes is a central question in biology. High-content phenotyping approaches such as Perturb-seq (single-cell RNA-sequencing pooled CRISPR screens) present an opportunity for exploring such genetic interactions (GIs) at scale. Here, we present an analytical framework for interpreting high-dimensional landscapes of cell states (manifolds) constructed from transcriptional phenotypes.

Mapping the Genetic Landscape of Human Cells.

Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs.

The regulatory protein SnoN antagonizes activin/Smad2 protein signaling and thereby promotes adipocyte differentiation and obesity in mice.

Ski-related oncogene SnoN (SnoN or SKIL) regulates multiple signaling pathways in a tissue- and developmental stage-dependent manner and has broad functions in embryonic angiogenesis, mammary gland alveologenesis, cancer, and aging. Here, we report that SnoN also plays a critical role in white adipose tissue (WAT) development by regulating mesenchymal stem cell (MSC) self-renewal and differentiation. We found that SnoN promotes MSC differentiation in the adipocyte lineage by antagonizing activin A/Smad2, but not TGFβ/Smad3 signaling.

SnoN Antagonizes the Hippo Kinase Complex to Promote TAZ Signaling during Breast Carcinogenesis.

SnoN regulates multiple signaling pathways, including TGF-β/Smad and p53, and displays both pro-oncogenic and anti-oncogenic activities in human cancer. We have observed previously that both its intracellular localization and expression levels are sensitive to cell density, suggesting that it may crosstalk with Hippo signaling. Here we report that, indeed, SnoN interacts with multiple components of the Hippo pathway to inhibit the binding of Lats2 to TAZ and the subsequent phosphorylation of TAZ, leading to TAZ stabilization.