CRISPR-Mediated Knockout of Long 3′ UTR mRNA Isoforms in mESC-Derived Neurons

Alternative cleavage and polyadenylation (APA) is pervasive, occurring for more than 70% of human and mouse genes. Distal poly(A) site selection to generate longer 3′ UTR mRNA isoforms is prevalent in the nervous system, affecting thousands of genes. Here, we establish mouse embryonic stem cell (mESC)-derived neurons (mES-neurons) as a suitable system to study long 3′ UTR isoforms. RNA-seq analysis revealed that mES-neurons show widespread 3′ UTR lengthening that closely resembles APA patterns found in mouse cortex. mESCs are highly amenable to genetic manipulation. We present a method to eliminate long 3′ UTR isoform expression using CRISPR/Cas9 editing. This approach can lead to clones with the desired deletion within several weeks. We demonstrate this strategy on the Mprip gene as a proof-of-principle. To confirm loss of long 3′ UTR expression and the absence of cryptic poly(A) site usage stemming from the CRISPR deletion, we present a simple and cost-efficient targeted long-read RNA-sequencing strategy using the Oxford Nanopore Technologies platform. Using this method, we confirmed specific loss of the Mprip long 3′ UTR isoform. CRISPR gene editing of mESCs thus serves as a highly relevant platform for studying the molecular and cellular functions of long 3′ UTR mRNA isoforms.

Understanding the source of METTL3-independent m6A in mRNA

N6-methyladenosine (m6A) is a highly prevalent mRNA modification which promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30—80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we re-examined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines which continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3's role in synthesizing m6A using a genomic deletion of Mettl3, and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

Cell-cell communication through FGF4 generates and maintains robust proportions of differentiated cell types in embryonic stem cells

During embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell type proportioning despite variable initial conditions in the precursor cells, as well as the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterised. Here we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range FGF4 signal. We characterise the molecular and dynamical properties of the communication mechanism, and show how it controls both robust cell type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signalling that may operate in a range of developing tissues.

Live cell imaging of metabolic heterogeneity by quantitative fluorescent ATP indicator protein, QUEEN-37C.

Adenosine triphosphate (ATP) is often referred as the energy currency of the cell. Yet, non-invasive, real-time, and quantitative measurement of its concentration in living mammalian cells has been difficult. Here we report an improved fluorescent ATP indicator protein, QUEEN-37C, which is optimized for measuring ATP concentration in living mammalian cells. Absolute value of the ATP concentration can be estimated from the ratiometric fluorescence imaging, and its accuracy was verified by the luciferase assay. Since QUEEN-37C enables the single-cell measurement of ATP concentration, we can not only measure its mean but its distribution in the cell population, which revealed that the ATP concentration is tightly regulated in most cells. We also noted the positive correlations in the ATP concentration among adjacent cells in epithelial cell sheet and mouse embryonic stem cell colonies. Thus, QUEEN-37C would serve as a new tool for the investigation of the single cell heterogeneity of metabolic states.

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

ABSTRACTRecent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.