RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance.

Regulatory T cells (Treg cells) can express the transcription factors T-bet and GATA-3, but the function of this expression and whether such cells represent stable subsets is still unknown. By using various reporter tools, we found that the expression of T-bet and GATA-3 in Treg cells was dynamically influenced by the cytokine environment. Treg cell-specific deletion of the gene encoding either T-bet (Tbx21) or GATA-3 (Gata3) alone did not result in loss of Treg cell function; however, mice with combined deficiency in both genes in Treg cells developed severe autoimmune-like diseases. Loss of Treg cell function correlated with upregulation of expression of the transcription factor RORγt and reduced expression of the transcription factor Foxp3. Thus, in the steady state, activated Treg cells transiently upregulated either T-bet or GATA-3 to maintain T cell homeostasis.

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A universal trade-off between growth and lag in fluctuating environments.

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.

Expression and regulation of intergenic long noncoding RNAs during T cell development and differentiation.

Although intergenic long noncoding RNAs (lincRNAs) have been linked to gene regulation in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identified 1,524 lincRNA clusters in 42 T cell samples, from early T cell progenitors to terminally differentiated helper T cell subsets. Our analysis revealed highly dynamic and cell-specific expression patterns for lincRNAs during T cell differentiation. These lincRNAs were located in genomic regions enriched for genes that encode proteins with immunoregulatory functions. Many were bound and regulated by the key transcription factors T-bet, GATA-3, STAT4 and STAT6. We found that the lincRNA LincR-Ccr2-5'AS, together with GATA-3, was an essential component of a regulatory circuit in gene expression specific to the TH2 subset of helper T cells and was important for the migration of TH2 cells.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

During post-implantation development of the mouse embryo, descendants of the inner cell mass in the early epiblast transit from the naive to primed pluripotent state1. Concurrently, germ layers are formed and cell lineages are specified, leading to the establishment of the blueprint for embryogenesis. Fate-mapping and lineage-analysis studies have revealed that cells in different regions of the germ layers acquire location-specific cell fates during gastrulation2-5. The regionalization of cell fates preceding the formation of the basic body plan-the mechanisms of which are instrumental for understanding embryonic programming and stem-cell-based translational study-is conserved in vertebrate embryos6-8. However, a genome-wide molecular annotation of lineage segregation and tissue architecture of the post-implantation embryo has yet to be undertaken. Here we report a spatially resolved transcriptome of cell populations at defined positions in the germ layers during development from pre- to late-gastrulation stages. This spatiotemporal transcriptome provides high-resolution digitized in situ gene-expression profiles, reveals the molecular genealogy of tissue lineages and defines the continuum of pluripotency states in time and space. The transcriptome further identifies the networks of molecular determinants that drive lineage specification and tissue patterning, supports a role of Hippo-Yap signalling in germ-layer development and reveals the contribution of visceral endoderm to the endoderm in the early mouse embryo.

Loss of the TRPM4 channel in humans causes immune dysregulation with defective monocyte migration.

BACKGROUND: TRPM4 is a broadly expressed, calcium-activated, monovalent cation channel that regulates immune cell function in mice and cell lines. Clinically, however, partial loss- or gain-of-function mutations in TRPM4 lead to arrhythmia and heart disease, with no documentation of immunologic disorders. OBJECTIVE: To characterize functional cellular mechanisms underlying the immune dysregulation phenotype in a proband with a mutated TRPM4 gene. METHODS: We employed a combination of biochemical, cell biological, imaging, omics analyses, flow cytometry, and gene editing approaches. RESULTS: We report the first human cases to our knowledge with complete loss of the TRPM4 channel, leading to immune dysregulation with frequent bacterial and fungal infections. Single-cell and bulk RNA sequencing point to altered expression of genes affecting cell migration, specifically in monocytes. Inhibition of TRPM4 in T cells and the THP-1 monocyte cell line reduces migration. More importantly, primary T cells and monocytes from TRPM4 patients migrate poorly. Finally, CRISPR knockout of TRPM4 in THP-1 cells greatly reduces their migration potential. CONCLUSION: Our results demonstrate that TRPM4 plays a critical role in regulating immune cell migration, leading to increased susceptibility to infections.

Methods for dynamic and whole volume imaging of the zebrafish heart.

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.