Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing.

mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

Observing dynamical phases of BCS superconductors in a cavity QED simulator.

In conventional Bardeen-Cooper-Schrieffer superconductors1, electrons with opposite momenta bind into Cooper pairs due to an attractive interaction mediated by phonons in the material. Although superconductivity naturally emerges at thermal equilibrium, it can also emerge out of equilibrium when the system parameters are abruptly changed2-8. The resulting out-of-equilibrium phases are predicted to occur in real materials and ultracold fermionic atoms, but not all have yet been directly observed. Here we realize an alternative way to generate the proposed dynamical phases using cavity quantum electrodynamics (QED). Our system encodes the presence or absence of a Cooper pair in a long-lived electronic transition in 88Sr atoms coupled to an optical cavity and represents interactions between electrons as photon-mediated interactions through the cavity9,10. To fully explore the phase diagram, we manipulate the ratio between the single-particle dispersion and the interactions after a quench and perform real-time tracking of the subsequent dynamics of the superconducting order parameter using nondestructive measurements. We observe regimes in which the order parameter decays to zero (phase I)3,4, assumes a non-equilibrium steady-state value (phase II)2,3 or exhibits persistent oscillations (phase III)2,3. This opens up exciting prospects for quantum simulation, including the potential to engineer unconventional superconductors and to probe beyond mean-field effects like the spectral form factor11,12, and for increasing the coherence time for quantum sensing.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

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

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Accumulation and ineffective silencing of transposable elements on an avian W Chromosome.

One of the defining features of transposable elements (TEs) is their ability to move to new locations in the host genome. To minimize the potentially deleterious effects of de novo TE insertions, hosts have evolved several mechanisms to control TE activity, including recombination-mediated removal and epigenetic silencing; however, increasing evidence suggests that silencing of TEs is often incomplete. The crow family experienced a recent radiation of LTR retrotransposons (LTRs), offering an opportunity to gain insight into the regulatory control of young, potentially still active TEs. We quantified the abundance of TE-derived transcripts across several tissues in 15 Eurasian crows (Corvus (corone) spp.) raised under common garden conditions and find evidence for ineffective TE suppression on the female-specific W Chromosome. Using RNA-seq data, we show that ∼9.5% of all transcribed TEs had considerably greater (average, 16-fold) transcript abundance in female crows and that >85% of these female-biased TEs originated on the W Chromosome. After accounting for differences in TE density among chromosomal classes, W-linked TEs were significantly more highly expressed than TEs residing on other chromosomes, consistent with ineffective silencing on the former. Together, our results suggest that the crow W Chromosome acts as a source of transcriptionally active TEs, with possible negative fitness consequences for female birds analogous to Drosophila (an X/Y system), in which overexpression of Y-linked TEs is associated with male-specific aging and fitness loss ("toxic Y").

Boron Nitride Nanosheets Induce Lipid Accumulation and Autophagy in Human Alveolar Lung Epithelial Cells Cultivated at Air-Liquid Interface.

Hexagonal boron nitride (hBN) is an emerging 2D material attracting significant attention due to its superior electrical, chemical, and therapeutic properties. However, inhalation toxicity mechanisms of hBN in human lung cells are poorly understood. Here, cellular interaction and effects of hBN nanosheets is investigated in alveolar epithelial cells cultured on porous inserts and exposed under air-liquid interface conditions for 24 h. hBN is taken up by the cells as determined in a label-free manner via RAMAN-confocal microscopy, ICP-MS, TEM, and SEM-EDX. No significant (p > 0.05) effects are observed on cell membrane integrity (LDH release), epithelial barrier integrity (TEER), interleukin-8 cytokine production or reactive oxygen production at tested dose ranges (1, 5, and 10 µg cm-2). However, it is observed that an enhanced accumulation of lipid granules in cells indicating the effect of hBN on lipid metabolism. In addition, it is observed that a significant (p < 0.05) and dose-dependent (5 and 10 µg cm-2) induction of autophagy in cells after exposure to hBN, potentially associated with the downstream processing and breakdown of excess lipid granules to maintain lipid homeostasis. Indeed, lysosomal co-localization of lipid granules supporting this argument is observed. Overall, the results suggest that the continuous presence of excess intracellular lipids may provoke adverse outcomes in the lungs.

Cathodoluminescent and Characteristic X-Ray-Emissive Rare-Earth-Doped Core/Shell Protein Labels for Spectromicroscopic Analysis of Cell Surface Receptors.

Understanding the localization and the interactions of biomolecules at the nanoscale and in the cellular context remains challenging. Electron microscopy (EM), unlike light-based microscopy, gives access to the cellular ultrastructure yet results in grey-scale images and averts unambiguous (co-)localization of biomolecules. Multimodal nanoparticle-based protein labels for correlative cathodoluminescence electron microscopy (CCLEM) and energy-dispersive X-ray spectromicroscopy (EDX-SM) are presented. The single-particle STEM-cathodoluminescence (CL) and characteristic X-ray emissivity of sub-20 nm lanthanide-doped nanoparticles are exploited as unique spectral fingerprints for precise label localization and identification. To maximize the nanoparticle brightness, lanthanides are incorporated in a low-phonon host lattice and separated from the environment using a passivating shell. The core/shell nanoparticles are then functionalized with either folic (terbium-doped) or caffeic acid (europium-doped). Their potential for (protein-)labeling is successfully demonstrated using HeLa cells expressing different surface receptors that bind to folic or caffeic acid, respectively. Both particle populations show single-particle CL emission along with a distinctive energy-dispersive X-ray signal, with the latter enabling color-based localization of receptors within swift imaging times well below 2 min per µ m $\umu\text{m}$ 2 while offering high resolution with a pixel size of 2.78 nm. Taken together, these results open a route to multi-color labeling based on electron spectromicroscopy.