Biology subjects

Stewart, E. M.

Publications and source records attributed to Stewart, E. M..

3 recordsLinked to original sources

bioRxiv2026

A high-resolution protein-RNA binding atlas for structure-aware interaction prediction and post-transcriptional regulatory modeling

RNA-binding proteins (RBPs) control post-transcriptional gene expression by regulating RNA processing, localization, stability, and translation. However, our understanding of RNA regulation remains limited by the resolution, scale, and uniformity of available RBP binding maps. Here we introduce eclair CLIP (ecCLIP), a high-throughput CLIP-seq platform that combines endogenous protein tagging for uniform affinity capture with ligation-free library preparation to generate high-resolution RNA-protein binding profiles. We applied ecCLIP to 67 endogenously tagged RBPs in HEK293T cells, generating a high-quality transcriptome-scale atlas that recovered known binding preferences and captured post-transcriptional regulons linked to RNA half-life. To test the utility of the atlas, we incorporated ecCLIP into Borzuki, a Borzoi-derived sequence-to-function model trained on complementary RNA degradation, expression, CLIP, and Perturb-seq data. Inclusion of ecCLIP data improved RNA half-life prediction and assessment of 3' UTR variant effects on RNA stability, demonstrating that our atlas helps capture RNA dynamics. To directly model the cis-regulatory grammar underlying these RBP-RNA interactions, we developed Pythia, a structure-aware neural network architecture that incorporates context-free grammar-inspired fixed convolutions to explicitly encode RNA base-pairing features. Pythia predicted RBP binding from sequence and structural context and decomposed predictions into sequence- and structure-dependent components. We tested this decomposition for five RBPs using massively parallel reporter CLIP (MPRC) binding assays in cells, using synthetic RNA libraries containing mutations designed to preserve or disrupt predicted RNA structures across nominated binding sites. We used the same MPRC strategy to confirm allele-specific binding effects for variants prioritized by Pythia. Together, ecCLIP and Pythia provide a resource for structure-aware decoding of RNA-protein interaction grammar and interpretation of post-transcriptional regulatory variation.

genomics↗

bioRxiv2025

Stag2 dependent chromatin remodeling enforces the erythroid-specific Gata1 cistrome

The transcription factor GATA1 has pleiotropic hematopoietic functions, particularly in erythroid and megakaryocytic ontogeny. While mechanistic investigations have uncovered many facets of GATA1 biology, how GATA1 co-regulates divergent cell fates remains only partially characterized. We previously described that loss of Stag2, a member of the cohesin complex and a recurrent mutational target in myelodysplastic syndrome and Down Syndrome associated acute megakaryoblastic leukemia, results in altered chromatin accessibility, transcription factor function, and cell differentiation. Hence, we hypothesized that chromatin accessibility facilitates lineage specificity of GATA1, thereby permitting efficient cellular differentiation. To understand the connection between chromatin accessibility and GATA1, we performed comprehensive studies of erythropoiesis in Stag2{Delta} mice. Defects in Stag2-deficient hematopoiesis included reduced numbers of erythroid progenitors (EryPs), impaired terminal erythroid maturation, increased number of MkPs, and increased megakaryocytes. RNA- and ATAC-sequencing of EryPs revealed altered patterns of Gata1 target gene expression with altered accessibility in conjunction with loss of expression of erythroid targets and gain of megakaryocyte targets. Gata1 occupancy was lost at erythroid targets, while occupancy increased at megakaryocyte targets. Functionally, we observed that Stag2-deficient EryPs have diminished erythroid output and augmented megakaryocyte output in orthogonal differentiation assays. Human models and primary MDS patients recapitulated the essential phenotypic and molecular features of our in vivo murine MDS model. Collectively, this study advances the understanding of the interplay between TF function and chromatin accessibility. Moreover, these data suggest a novel conceptual paradigm of dyserythropoiesis in MDS. Key Pointsxxxxx

molecular biology↗

bioRxiv2019

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

genomics↗