Cheng, A. Y. | Search bioRxiv

bioRxiv2025

RNA structural heterogeneity in a eukaryotic cell influences its heat shock response

RNA structure-based gene regulation remains under-explored in eukaryotes. While RNA can form different conformations in solution, the extent to which it folds into structure ensembles and how the different conformations regulate gene expression still needs to be fully understood. We coupled the SHAPE compound NAI-N3 with direct RNA sequencing to identify structure modifications along a single RNA molecule (sm-PORE-cupine). Using a combination of base mapping and direct signal alignment, we boosted the percentage of mappable RNA molecules from direct RNA sequencing. Using Bernoulli Mixture Model (BMM) clustering, we show that we can separate RNA structure ensembles from ligand-bound and unbound riboswitches accurately, identify isoform-specific structure ensembles along the SARS-CoV-2 genome, and determine RNA structure ensembles in the transcriptome of a eukaryote, C. albicans, at yeast (30{degrees}C) and hyphae (37{degrees}C) states. We observed that RNAs are more structurally homogenous at 37{degrees}C compared to 30{degrees}C, are more variable in vivo than in vitro, and show higher homogeneity in 3UTRs than in the coding region. We also identified structure ensembles that are associated with changes in translation efficiency and decay in C. albicans at 30{degrees}C and 37{degrees}C and validated translational changes using reporter assays. Our work shows that single-molecule RNA structure probing using direct RNA sequencing can be applied to diverse transcriptomes to study the complexity and function of RNA structures.

molecular biology↗

bioRxiv2024

In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

Chimeric antigen receptor (CAR) T cells are highly effective in hematologic malignancies. However, loss of CAR T cells can contribute to relapse in a significant number of patients. These limitations could potentially be overcome by targeted gene editing to increase CAR T cell persistence. Here, we performed in vivo loss-of-function CRISPR screens in BCMA-targeting CAR T cells to investigate genes that influence CAR T cell persistence, function and efficacy in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR-library edited T cells in vitro and then at early and late timepoints in vivo to track the performance of gene modified CAR T cells from manufacturing to survival in tumors. The screens revealed several context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, while loss of PTPN2, ZC3H12A, and RC3H1 conferred early selective growth advantages to CAR T cells in vivo. Strikingly, we identified cyclin-dependent kinase inhibitor 1B (CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late timepoints in vivo. CDKN1B ablation increased BCMA CAR T cell proliferation and effector function in response to antigen, significantly enhancing tumor clearance and overall survival. Thus, our findings reveal differing effects of gene-perturbation on CAR T cells over time and in different selective environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma, and underscore the importance of in vivo screening as a tool for identifying genes to enhance CAR T cell function and efficacy.

immunology↗