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In this article, 3 models of collaboration between speech-language pathologists and classroom teachers are discussed to promote emergent literacy and accurate and fluent word recognition. These models are demonstration lessons, team teaching, and consultation. A number of instructional principles are presented for emergent literacy and decoding within the context of collaborative work designed to help a preschool child with oral language difficulties and a second grader with word reading problems. Instructional principles cover the areas of vocabulary, phonological awareness, and narrative discourse in the emergent literacy period, and sounding out, reading by analogy, structural analysis, and routines to build fluency during the period of formal reading instruction. [ABSTRACT FROM AUTHOR]

Narrative writing is a complex process that has its basis in oral narration. The development of the story form is described, and approaches to the study of narration are reviewed. The written narrative patterns of students with learning disabilities are detailed along with a discussion of the reasons for their difficulties with this genre of writing. Instructional approaches are presented that have been effective in improving the written narrative skills of these youngsters. Copyright © 2000 by Aspen Publishers, Inc. [ABSTRACT FROM AUTHOR]

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease causing. This creates a new bottleneck: determining the functional impact of each variant-typically a painstaking, customized process undertaken one or a few genes and variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,448 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of uncertain significance. Our publicly available resource extends our understanding of coding variation in human diseases., Competing Interests: Declaration of interests A.E.C. serves as a scientific advisor for Recursion, Quiver, and SyzOnc, which use image-based profiling for drug discovery, and receives honoraria for occasional talks at pharmaceutical and biotechnology companies. F.P.R. is a scientific advisor and investor in Constantiam Biosciences, which provides tools for clinical variant annotation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Legionella pneumophila uses over 300 translocated effector proteins to rewire host cells during infection and create a replicative niche for intracellular growth. To date, several studies have identified L. pneumophila effectors that indirectly and directly regulate the activity of other effectors, providing an additional layer of regulatory complexity. Among these are "metaeffectors," a special class of effectors that regulate the activity of other effectors once inside the host. A defining feature of metaeffectors is direct, physical interaction with a target effector. Metaeffector identification, to date, has depended on phenotypes in heterologous systems and experimental serendipity. Using a multiplexed, recombinant barcode-based yeast two-hybrid technology we screened for protein-protein interactions among all L. pneumophila effectors and 28 components of the Dot/Icm type IV secretion system (>167,000 protein combinations). Of the 52 protein interactions identified by this approach, 44 are novel protein interactions, including 10 novel effector-effector interactions (doubling the number of known effector-effector interactions)., Importance: Secreted bacterial effector proteins are typically viewed as modulators of host activity, entering the host cytosol to physically interact with and modify the activity of one or more host proteins in support of infection. A growing body of evidence suggests that a subset of effectors primarily function to modify the activities of other effectors inside the host. These "effectors of effectors" or metaeffectors are often identified through experimental serendipity during the study of canonical effector function against the host. We previously performed the first global effector-wide genetic interaction screen for metaeffectors within the arsenal of Legionella pneumophila , an intracellular bacterial pathogen with over 300 effectors. Here, using a high-throughput, scalable methodology, we present the first global interaction network of physical interactions between L. pneumophila effectors. This data set serves as a complementary resource to identify and understand both the scope and nature of non-canonical effector activity within this important human pathogen.

Malate is an important dicarboxylic acid produced from fumarate in the tricarboxylic acid cycle. Deficiencies of fumarate hydrolase (FH) and malate dehydrogenase (MDH), responsible for malate formation and metabolism, respectively, are known to cause recessive forms of neurodevelopmental disorders (NDDs). The malic enzyme isoforms, malic enzyme 1 (ME1) and 2 (ME2), are required for the conversion of malate to pyruvate. To date, there have been no reports linking deficiency of either malic enzyme isoforms to any Mendelian disease in humans. We report a patient presenting with NDD, subtle dysmorphic features, resolved dilated cardiomyopathy, and mild blood lactate elevation. Whole exome sequencing (WES) revealed a homozygous frameshift variant (c.1379_1380delTT, p.Phe460fs*22) in the malic enzyme 2 (ME2) gene resulting in truncated and unstable ME2 protein in vitro. Subsequent deletion of the yeast ortholog of human ME2 (hME2) resulted in growth arrest, which was rescued by overexpression of hME2, strongly supporting an important role of ME2 in mitochondrial function. Our results also support the pathogenicity and candidacy of the ME2 gene and variant in association with NDD. To our knowledge, this is the first report of a Mendelian human disease resulting from a biallelic variant in the ME encoding gene. Future studies are warranted to confirm ME2-associated recessive NDD., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This paper presents an evaluation of predictions submitted for the "HMBS" challenge, a component of the sixth round of the Critical Assessment of Genome Interpretation held in 2021. The challenge required participants to predict the effects of missense variants of the human HMBS gene on yeast growth. The HMBS enzyme, critical for the biosynthesis of heme in eukaryotic cells, is highly conserved among eukaryotes. Despite the application of a variety of algorithms and methods, the performance of predictors was relatively similar, with Kendall's tau correlation coefficients between predictions and experimental scores around 0.3 for a majority of submissions. Notably, the median correlation (≥ 0.34) observed among these predictors, especially the top predictions from different groups, was greater than the correlation observed between their predictions and the actual experimental results. Most predictors were moderately successful in distinguishing between deleterious and benign variants, as evidenced by an area under the receiver operating characteristic (ROC) curve (AUC) of approximately 0.7 respectively. Compared with the recent two rounds of CAGI competitions, we noticed more predictors outperformed the baseline predictor, which is solely based on the amino acid frequencies. Nevertheless, the overall accuracy of predictions is still far short of positive control, which is derived from experimental scores, indicating the necessity for considerable improvements in the field. The most inaccurately predicted variants in this round were associated with the insertion loop, which is absent in many orthologs, suggesting the predictors still heavily rely on the information from multiple sequence alignment., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Computational variant effect predictors offer a scalable and increasingly reliable means of interpreting human genetic variation, but concerns of circularity and bias have limited previous methods for evaluating and comparing predictors. Population-level cohorts of genotyped and phenotyped participants that have not been used in predictor training can facilitate an unbiased benchmarking of available methods. Using a curated set of human gene-trait associations with a reported rare-variant burden association, we evaluate the correlations of 24 computational variant effect predictors with associated human traits in the UK Biobank and All of Us cohorts., Results: AlphaMissense outperformed all other predictors in inferring human traits based on rare missense variants in UK Biobank and All of Us participants. The overall rankings of computational variant effect predictors in these two cohorts showed a significant positive correlation., Conclusion: We describe a method to assess computational variant effect predictors that sidesteps the limitations of previous evaluations. This approach is generalizable to future predictors and could continue to inform predictor choice for personal and clinical genetics., (© 2024. The Author(s).)

Multiplexed assays of variant effect (MAVEs) have emerged as a powerful approach for interrogating thousands of genetic variants in a single experiment. The flexibility and widespread adoption of these techniques across diverse disciplines have led to a heterogeneous mix of data formats and descriptions, which complicates the downstream use of the resulting datasets. To address these issues and promote reproducibility and reuse of MAVE data, we define a set of minimum information standards for MAVE data and metadata and outline a controlled vocabulary aligned with established biomedical ontologies for describing these experimental designs., (© 2024. The Author(s).)

Background: Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms underlying variant effects in human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity., Results: Using a yeast growth-based assay, we score the abundance of 95% of GCK missense and nonsense variants. When combining the abundance scores with our previously determined activity scores, we find that 43% of hypoactive variants also decrease cellular protein abundance. The low-abundance variants are enriched in the large domain, while residues in the small domain are tolerant to mutations with respect to abundance. Instead, many variants in the small domain perturb GCK conformational dynamics which are essential for appropriate activity., Conclusions: In this study, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis., (© 2024. The Author(s).)

Motivation: Long-read sequencing technologies, an attractive solution for many applications, often suffer from higher error rates. Alignment of multiple reads can improve base-calling accuracy, but some applications, e.g. sequencing mutagenized libraries where multiple distinct clones differ by one or few variants, require the use of barcodes or unique molecular identifiers. Unfortunately, sequencing errors can interfere with correct barcode identification, and a given barcode sequence may be linked to multiple independent clones within a given library., Results: Here we focus on the target application of sequencing mutagenized libraries in the context of multiplexed assays of variant effects (MAVEs). MAVEs are increasingly used to create comprehensive genotype-phenotype maps that can aid clinical variant interpretation. Many MAVE methods use long-read sequencing of barcoded mutant libraries for accurate association of barcode with genotype. Existing long-read sequencing pipelines do not account for inaccurate sequencing or nonunique barcodes. Here, we describe Pacybara, which handles these issues by clustering long reads based on the similarities of (error-prone) barcodes while also detecting barcodes that have been associated with multiple genotypes. Pacybara also detects recombinant (chimeric) clones and reduces false positive indel calls. In three example applications, we show that Pacybara identifies and correctly resolves these issues., Availability and Implementation: Pacybara, freely available at https://github.com/rothlab/pacybara, is implemented using R, Python, and bash for Linux. It runs on GNU/Linux HPC clusters via Slurm, PBS, or GridEngine schedulers. A single-machine simplex version is also available., (© The Author(s) 2024. Published by Oxford University Press.)

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants., Competing Interests: Declaration of interests F.P.R. is an investor in Ranomics, Inc., and is an investor in and advisor for SeqWell, Inc., BioSymetrics, Inc., and Constantiam Biosciences, Inc., and has accepted conference travel support from Illumina, Inc. L.F., A.C., and R.N. are employed by and invested in Invitae. R.J.D. has received both a grant and royalties and has also served as a consultant for Alnylam Pharmaceuticals., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance., Competing Interests: Declaration of interests D.D. is a shareholder and advisor for Repare Therapeutics. A.-C.G. is a member of Molecular Cell’s Advisory Board., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The COVID-19 pandemic has catalyzed unprecedented scientific data and reagent sharing and collaboration, which enabled understanding the virology of the SARS-CoV-2 virus and vaccine development at record speed. The pandemic, however, has also raised awareness of the danger posed by the family of coronaviruses, of which 7 are known to infect humans and dozens have been identified in reservoir species, such as bats, rodents, or livestock. To facilitate understanding the commonalities and specifics of coronavirus infections and aspects of viral biology that determine their level of lethality to the human host, we have generated a collection of freely available clones encoding nearly all human coronavirus proteins known to date. We hope that this flexible, Gateway-compatible vector collection will encourage further research into the interactions of coronaviruses with their human host, to increase preparedness for future zoonotic viral outbreaks., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Background: Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants., Result: Here, we exploit a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlate with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants are concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation., Conclusion: Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK., (© 2023. The Author(s).)

The impact of millions of individual genetic variants on molecular phenotypes in coding sequences remains unknown. Multiplexed assays of variant effect (MAVEs) are scalable methods to annotate relevant variants, but existing software lacks standardization, requires cumbersome configuration, and does not scale to large targets. We present satmut_utils as a flexible solution for simulation and variant quantification. We then benchmark MAVE software using simulated and real MAVE data. We finally determine mRNA abundance for thousands of cystathionine beta-synthase variants using two experimental methods. The satmut_utils package enables high-performance analysis of MAVEs and reveals the capability of variants to alter mRNA abundance., (© 2023. The Author(s).)

Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function., (© 2023. The Author(s).)

Understanding the mechanisms of coronavirus disease 2019 (COVID-19) disease severity to efficiently design therapies for emerging virus variants remains an urgent challenge of the ongoing pandemic. Infection and immune reactions are mediated by direct contacts between viral molecules and the host proteome, and the vast majority of these virus-host contacts (the 'contactome') have not been identified. Here, we present a systematic contactome map of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the human host encompassing more than 200 binary virus-host and intraviral protein-protein interactions. We find that host proteins genetically associated with comorbidities of severe illness and long COVID are enriched in SARS-CoV-2 targeted network communities. Evaluating contactome-derived hypotheses, we demonstrate that viral NSP14 activates nuclear factor κB (NF-κB)-dependent transcription, even in the presence of cytokine signaling. Moreover, for several tested host proteins, genetic knock-down substantially reduces viral replication. Additionally, we show for USP25 that this effect is phenocopied by the small-molecule inhibitor AZ1. Our results connect viral proteins to human genetic architecture for COVID-19 severity and offer potential therapeutic targets., (© 2022. The Author(s).)

Scalable sequence-function studies have enabled the systematic analysis and cataloging of hundreds of thousands of coding and noncoding genetic variants in the human genome. This has improved clinical variant interpretation and provided insights into the molecular, biophysical, and cellular effects of genetic variants at an astonishing scale and resolution across the spectrum of allele frequencies. In this review, we explore current applications and prospects for the field and outline the principles underlying scalable functional assay design, with a focus on the study of single-nucleotide coding and noncoding variants.

SARS-CoV-2 viral infection led to the devastating COVID-19 pandemic, where illness stemmed from interactions between virions and recipient host cells resulting in multi-layered pathological consequences. The role of the infection portal is now understood to be the cellular angiotensin converting enzyme-2 (ACE2) receptor, which binds to viral spike (S) protein initiating virion internalisation process. Since SARS-CoV-2 virions bear some resemblance to endogenously produced small extracellular vesicles (sEVs) we reasoned that EVs engineered to express S protein (viral mimics) may interfere with viral infection. Here, we report generation of HEK293T cells producing sEVs enriched for transmembrane S-protein tagged with green fluorescent protein (S/GFP). Strikingly, S protein drove the GFP tag to the membrane of sEVs, while GFP alone was not efficiently included in the sEV cargo. High-throughput quantitative proteomics revealed that S/GFP sEVs contained over 1000 proteins including canonical components of the exosomal pathway such as ALIX, syntenin-1, and tetraspanins (CD81, CD9), but depleted for calnexin and cytochrome c. We found that 84 sEV proteins were significantly altered by the presence of S/GFP. S protein expressing EVs efficiently adhered to target cells in an ACE2-dependent manner, but they were poorly internalised. Importantly, prolonged administration of S/GFP EV to K18-hACE2 mice provided a significant protection against SARS-CoV-2 infection. Thus, the generation of sEV containing S protein can be considered as a novel therapeutic approach in reducing the transmission of SARS-CoV-2., Competing Interests: The authors report no conflicts of interest., (© 2022 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Summary: Multiplexed assays of variant effect (MAVEs) are capable of experimentally testing all possible single nucleotide or amino acid variants in selected genomic regions, generating 'variant effect maps', which provide biochemical insight and functional evidence to enable more rapid and accurate clinical interpretation of human variation. Because the international community applying MAVE approaches is growing rapidly, we developed the online MaveRegistry platform to catalyze collaboration, reduce redundant efforts, allow stakeholders to nominate targets and enable tracking and sharing of progress on ongoing MAVE projects., Availability and Implementation: MaveRegistry service: https://registry.varianteffect.org. MaveRegistry source code: https://github.com/kvnkuang/maveregistry-front-end., (© The Author(s) 2021. Published by Oxford University Press.)

Systematic, high-throughput screening for "dominant-negative" protein fragments is an emerging method for mapping functional regions of the parental protein in vivo. In this issue of Cell Systems, Ford et al. apply this approach to 65 cancer drivers, providing functional insights and demonstrating therapeutic potential for several dominant-negative peptides., Competing Interests: Declaration of interests F.P.R. advises and holds shares in SeqWell, Inc., BioSymetrics, Inc., and Constantiam Biosciences, Inc. and is a Ranomics, Inc. shareholder., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Motivation: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variants of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation., Results: Here, we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes (i) by the number of unique missense VUS that had been reported to ClinVar; (ii) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS and (iii) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation toward greater impact on clinical variant interpretation., Availability and Implementation: Source code available at: https://github.com/rothlab/mave-gene-prioritization., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4 , KIF11 , mTOR , PKM2 , SGPP1 , TTK , and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1 - TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kim et al.)

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it., (Copyright © 2020 Kim et al.)

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease., Competing Interests: MC, NR, JS, KS, KM, MV, SS, FR, DD, DM, AR, DF No competing interests declared, (© 2020, Chiasson et al.)

Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Summary: Fully realizing the promise of personalized medicine will require rapid and accurate classification of pathogenic human variation. Multiplexed assays of variant effect (MAVEs) can experimentally test nearly all possible variants in selected gene targets. Planning a MAVE study involves identifying target genes with clinical impact, and identifying scalable functional assays for that target. Here, we describe MaveQuest, a web-based resource enabling systematic variant effect mapping studies by identifying potential functional assays, disease phenotypes and clinical relevance for nearly all human protein-coding genes., Availability and Implementation: MaveQuest service: https://mavequest.varianteffect.org/. MaveQuest source code: https://github.com/kvnkuang/mavequest-front-end/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Kinases are critical intracellular signaling proteins. To better understand kinase-mediated signal transduction, a large-scale human-yeast genetic interaction screen was performed. Among 597 human kinase genes tested, 28 displayed strong toxicity in yeast when overexpressed. En masse transformation of these toxic kinase genes into 4653 homozygous diploid yeast deletion mutants followed by barcode sequencing identified yeast toxicity modifiers and thus their human orthologs. Subsequent network analyses and functional grouping revealed that the 28 kinases and their 676 interaction partners (corresponding to a total of 969 genetic interactions) are enriched in cell death and survival (34%), small-molecule biochemistry (18%) and molecular transport (11%), among others. In the subnetwork analyses, a few kinases were commonly associated with glioma, cell migration and cell death/survival. Our analysis enabled the creation of a first draft of the kinase genetic interactome network and identified multiple drug targets for inflammatory diseases and cancer, in which deregulated kinase signaling plays a pathogenic role.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships 1,2 . Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome 3 , transcriptome 4 and proteome 5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.

Background: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective., Methods: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function., Results: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B 6 (ϱ = 0.93)., Conclusions: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.

Many traits are complex, depending non-additively on variant combinations. Even in model systems, such as the yeast S. cerevisiae, carrying out the high-order variant-combination testing needed to dissect complex traits remains a daunting challenge. Here, we describe "X-gene" genetic analysis (XGA), a strategy for engineering and profiling highly combinatorial gene perturbations. We demonstrate XGA on yeast ABC transporters by engineering 5,353 strains, each deleted for a random subset of 16 transporters, and profiling each strain's resistance to 16 compounds. XGA yielded 85,648 genotype-to-resistance observations, revealing high-order genetic interactions for 13 of the 16 transporters studied. Neural networks yielded intuitive functional models and guided exploration of fluconazole resistance, which was influenced non-additively by five genes. Together, our results showed that highly combinatorial genetic perturbation can functionally dissect complex traits, supporting pursuit of analogous strategies in human cells and other model systems., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiplex assays of variant effect (MAVEs), such as deep mutational scans and massively parallel reporter assays, test thousands of sequence variants in a single experiment. Despite the importance of MAVE data for basic and clinical research, there is no standard resource for their discovery and distribution. Here, we present MaveDB ( https://www.mavedb.org ), a public repository for large-scale measurements of sequence variant impact, designed for interoperability with applications to interpret these datasets. We also describe the first such application, MaveVis, which retrieves, visualizes, and contextualizes variant effect maps. Together, the database and applications will empower the community to mine these powerful datasets.