Assembly of a GPCR-G Protein Complex.

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch.

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2ß2) disorders, sickle cell disease, and ß-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to ß- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation.

Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers.

Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells.

How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only ∼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.

Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells.

Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.

Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces.

Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.

scATAC-seq preprocessing and imputation evaluation system for visualization, clustering and digital footprinting.

Single-cell ATAC-seq (scATAC-seq) is a recently developed approach that provides means to investigate open chromatin at single cell level, to assess epigenetic regulation and transcription factors binding landscapes. The sparsity of the scATAC-seq data calls for imputation. Similarly, preprocessing (filtering) may be required to reduce computational load due to the large number of open regions. However, optimal strategies for both imputation and preprocessing have not been yet evaluated together. We present SAPIEnS (scATAC-seq Preprocessing and Imputation Evaluation System), a benchmark for scATAC-seq imputation frameworks, a combination of state-of-the-art imputation methods with commonly used preprocessing techniques. We assess different types of scATAC-seq analysis, i.e. clustering, visualization and digital genomic footprinting, and attain optimal preprocessing-imputation strategies. We discuss the benefits of the imputation framework depending on the task and the number of the dataset features (peaks). We conclude that the preprocessing with the Boruta method is beneficial for the majority of tasks, while imputation is helpful mostly for small datasets. We also implement a SAPIEnS database with pre-computed transcription factor footprints based on imputed data with their activity scores in a specific cell type. SAPIEnS is published at: https://github.com/lab-medvedeva/SAPIEnS. SAPIEnS database is available at: https://sapiensdb.com.

Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters.

Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

Arabidopsis mRNA decay landscape shaped by XRN 5'-3' exoribonucleases.

5'-3' exoribonucleases (XRNs) play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of Arabidopsis wild-type and mutant plants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 activity but not nuclear XRN2 activity greatly altered Arabidopsis mRNA decay profiles. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the unique function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that pre-mRNA 3' end cleavage frequently occurs after adenosine. The most abundant decay intermediates in wild-type plants include not only the primary substrates of XRN4 but also the products of XRN4-mediated cytoplasmic decay. An increase in decay intermediates with 5' ends upstream of a consensus motif in the xrn4 mutant suggests that there is an endonucleolytic cleavage mechanism targeting the 3' untranslated regions of many Arabidopsis mRNAs. However, analysis of decay fragments in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence rarely result in major decay intermediates. Together, these findings reveal the major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.

Studying transcription factor function in the genome at molecular resolution.

About 7% of the human genome encodes cis-regulatory elements (CREs) that function as regulatory switches to modulate the expression of genes. These short genetic sequences control the complex transcriptional changes necessary for organismal development. A topical challenge in the field is to understand how transcription factors (TFs) read and translate this information into gene expression patterns. Here, I review how the development of single-molecule footprinting (SMF) that resolves the genome occupancy of TFs on individual DNA molecules resolution contributes to our ability to establish how the regulatory genetic information is interpreted at the mechanistic level. I further discuss how future developments in the nascent field of single-molecule genomics (SMG) could impact our understanding of gene regulation mechanisms.

Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B12-btuB Riboswitch Interaction.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.

Bottom-up global biodiversity metrics needed for businesses to assess and manage their impact.

Ensuring that companies can assess and manage their impacts on biodiversity will be crucial to solving the current biodiversity crisis, and regulatory and public pressure to disclose these impacts is increasing. Top-down intactness metrics (e.g., Mean Species Abundance) can be valuable for generating high-level or first-tier assessments of impact risk but do not provide sufficient precision or guidance for companies, regulators, or third-party assessors. New metrics based on bottom-up assessments of biodiversity (e.g., the Species Threat Abatement and Restoration metric) can accommodate spatial variation of biodiversity and provide more specific guidance for actions to avoid, reduce, remediate, and compensate for impacts and to identify positive opportunities.

Cuantificación vertical de la biodiversidad mundial necesarias para que las empresas evalúen y gestionen su impacto Resumen Para resolver la actual crisis de biodiversidad, es importante asegurar que las empresas puedan evaluar y gestionar su impacto sobre la biodiversidad. Además, cada vez es mayor la presión pública y legislativa para divulgar este impacto. La cuantificación vertical de la integridad (p. ej.: Abundancia Media de Especies) puede ser valiosa para producir evaluaciones de alto nivel o primera categoría del riesgo de impacto, pero no proporcionan suficiente precisión o guía para las empresas, los reguladores o los asesores de terceros. Las nuevas medidas basadas en evaluaciones verticales (p. ej.: la medida de Abatimiento y Restauración de Amenazas de Especies) pueden acomodar la variación espacial de la biodiversidad y proporcionar una guía más específica para las acciones necesarias para evitar, reducir, remediar y compensar los impactos e identificar las oportunidades positivas.

RGT: a toolbox for the integrative analysis of high throughput regulatory genomics data.

BACKGROUND: Massive amounts of data are produced by combining next-generation sequencing with complex biochemistry techniques to characterize regulatory genomics profiles, such as protein-DNA interaction and chromatin accessibility. Interpretation of such high-throughput data typically requires different computation methods. However, existing tools are usually developed for a specific task, which makes it challenging to analyze the data in an integrative manner. RESULTS: We here describe the Regulatory Genomics Toolbox (RGT), a computational library for the integrative analysis of regulatory genomics data. RGT provides different functionalities to handle genomic signals and regions. Based on that, we developed several tools to perform distinct downstream analyses, including the prediction of transcription factor binding sites using ATAC-seq data, identification of differential peaks from ChIP-seq data, and detection of triple helix mediated RNA and DNA interactions, visualization, and finding an association between distinct regulatory factors. CONCLUSION: We present here RGT; a framework to facilitate the customization of computational methods to analyze genomic data for specific regulatory genomics problems. RGT is a comprehensive and flexible Python package for analyzing high throughput regulatory genomics data and is available at: https://github.com/CostaLab/reg-gen . The documentation is available at: https://reg-gen.readthedocs.io.

Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

MALDI Peptide Mapping for Fast Analysis in Protein Footprinting.

Although protein footprinting results are commonly obtained by ESI-based LC-MS/MS, a more rapid-turnaround alternative approach is desirable to expand the scope of protein footprinting and facilitate routine analysis such as monitoring protein high order structure in quality control or checking epitope maps. Considering that MALDI is a faster procedure that can be easily adapted for high-throughput analysis, we explore here the feasibility of developing a MALDI-based analysis "portfolio" of bottom-up peptide mass mapping for footprinting. The approach was applied to several model proteins that were submitted to two footprinting strategies, FPOP and GEE labeling, and their performance was evaluated. We found adequate coverage that can be improved with automatic off-line separation and spotting, demonstrating the capability to footprint accurately protein conformational change, showing that MALDI may be useful for selected applications in protein footprinting.

Rewiring the specificity of extracytoplasmic function sigma factors.

Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

The Role of Virtual Consulting in Developing Environmentally Sustainable Health Care: Systematic Literature Review.

BACKGROUND: Health systems globally need to rapidly set and achieve targets for reaching net zero carbon emissions. Virtual consulting (including video- and telephone-based consulting) is regarded as one means by which this might be achieved, largely through reduced patient travel. Little is currently known about the ways in which forms of virtual consulting might contribute to the net zero agenda or how countries may develop and implement programs at scale that can support increased environmental sustainability. OBJECTIVE: In this paper, we asked, What is the impact of virtual consulting on environmental sustainability in health care? and What can we learn from current evaluations that can inform future reductions in carbon emissions? METHODS: We conducted a systematic review of published literature according to PRISMA (Preferred Reporting Item for Systematic Reviews and Meta-Analyses) guidelines. We searched the MEDLINE, PubMed, and Scopus databases using key terms relating to "carbon footprint," "environmental impact," "telemedicine," and "remote consulting," using citation tracking to identify additional articles. The articles were screened, and full texts that met the inclusion criteria were obtained. Data on the approach to carbon footprinting reported reductions in emissions, and the opportunities and challenges associated with the environmental sustainability of virtual consultations were extracted into a spreadsheet, analyzed thematically, and theorized using the Planning and Evaluating Remote Consultation Services framework to consider the various interacting influences, including environmental sustainability, that shape the adoption of virtual consulting services. RESULTS: A total of 1672 papers were identified. After removing duplicates and screening for eligibility, 23 papers that focused on a range of virtual consulting equipment and platforms across different clinical conditions and services were included. The focus on the environmental sustainability potential of virtual consulting was unanimously reported through carbon savings achieved by a reduction in travel related to face-to-face appointments. The shortlisted papers used a range of methods and assumptions to determine carbon savings, reporting these using different units and across varied sample sizes. This limited the potential for comparison. Despite methodological inconsistencies, all papers concluded that virtual consulting significantly reduced carbon emissions. However, there was limited consideration of wider factors (eg, patient suitability, clinical indication, and organizational infrastructure) influencing the adoption, use, and spread of virtual consultations and the carbon footprint of the entire clinical pathway in which the virtual consultation was provided (eg, risk of missed diagnoses from virtual consultations that result in the need for subsequent in-person consultations or admissions). CONCLUSIONS: There is overwhelming evidence that virtual consulting can reduce health care carbon emissions, largely through reducing travel related to in-person appointments. However, the current evidence fails to look at system factors associated with implementing virtual health care delivery and wider research into carbon emissions across the entire clinical pathway.

Advances in mass spectrometry-based footprinting of membrane proteins.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo-electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS-based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

Cancer Conformational Landscape Shapes Tumorigenesis.

During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of mutated proteins has been studied with atomic resolution, the precise impact of somatic mutations on the 3D proteome during malignant transformation remains unknown because methods to reveal in vivo protein structures in high throughput are limited. Here, we measured the accessibility of the lysine ε-amine for chemical modification across proteomes using covalent protein painting (CPP) to indirectly determine alterations in the 3D proteome. CPP is a novel, high-throughput quantitative mass spectrometric method that surveyed a total of 8052 lysine sites across the 60 cell lines of the well-studied anticancer cell line panel (NCI60). Overall, 5.2 structural alterations differentiated any cancer cell line from the other 59. Structural aberrations in 98 effector proteins correlated with the selected presence of 90 commonly mutated proteins in the NCI60 cell line panel, suggesting that different tumor genotypes reshape a limited set of effector proteins. We searched our dataset for druggable conformational aberrations and identified 49 changes in the cancer conformational landscape that correlated with the growth inhibition profiles of 300 drug candidates out of 50,000 small molecules. We found that alterations in heat shock proteins are key predictors of anticancer drug efficacy, which implies that the proteostasis network may have a general but hitherto unrecognized role in maintaining malignancy. Individual lysine sites may serve as biomarkers to guide drug selection or may be directly targeted for anticancer drug development.