Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Correction: An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing.

[This corrects the article DOI: 10.1371/journal.ppat.1008307.].

Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors.

Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2.

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

Global detection of human variants and isoforms by deep proteome sequencing.

An average shotgun proteomics experiment detects approximately 10,000 human proteins from a single sample. However, individual proteins are typically identified by peptide sequences representing a small fraction of their total amino acids. Hence, an average shotgun experiment fails to distinguish different protein variants and isoforms. Deeper proteome sequencing is therefore required for the global discovery of protein isoforms. Using six different human cell lines, six proteases, deep fractionation and three tandem mass spectrometry fragmentation methods, we identify a million unique peptides from 17,717 protein groups, with a median sequence coverage of approximately 80%. Direct comparison with RNA expression data provides evidence for the translation of most nonsynonymous variants. We have also hypothesized that undetected variants likely arise from mutation-induced protein instability. We further observe comparable detection rates for exon-exon junction peptides representing constitutive and alternative splicing events. Our dataset represents a resource for proteoform discovery and provides direct evidence that most frame-preserving alternatively spliced isoforms are translated.

Survival-based CRISPR genetic screens across a panel of permissive cell lines identify common and cell-specific SARS-CoV-2 host factors.

SARS-CoV-2 depends on host cell components for infection and replication. Identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection and replication. If druggable, host factor dependencies may present an attractive strategy for anti-viral therapy. In this study, we performed genome wide CRISPR knockout screens in Vero E6 cells and four human cell lines including Calu-3, UM-UC-4, HEK-293 and HuH-7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while other host genes identified were largely cell line specific, including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, immune-related pathways, and chromatin modification. Notably, the chromatin modifier gene KMT2C in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

Definition of germ layer cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate.

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.

Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns.

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.

SARS-CoV-2 nucleocapsid protein binds host mRNAs and attenuates stress granules to impair host stress response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is essential for viral replication, making it a promising target for antiviral drug and vaccine development. SARS-CoV-2 infected patients exhibit an uncoordinated immune response; however, the underlying mechanistic details of this imbalance remain obscure. Here, starting from a functional proteomics workflow, we cataloged the protein-protein interactions of SARS-CoV-2 proteins, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N localizes to stress granules and sequesters G3BPs away from their typical interaction partners, thus attenuating stress granule formation. We found that N binds directly to host mRNAs in cells, with a preference for 3' UTRs, and modulates target mRNA stability. We show that the N protein rewires the G3BP1 mRNA-binding profile and suppresses the physiological stress response of host cells, which may explain the imbalanced immune response observed in SARS-CoV-2 infected patients.

Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.

The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.

A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2.

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

Differential contribution of transcriptomic regulatory layers in the definition of neuronal identity.

Previous transcriptomic profiling studies have typically focused on separately analyzing mRNA expression, alternative splicing and alternative polyadenylation differences between cell and tissue types. However, the relative contribution of these three transcriptomic regulatory layers to cell type specification is poorly understood. This question is particularly relevant to neurons, given their extensive heterogeneity associated with brain location, morphology and function. In the present study, we generated profiles for the three regulatory layers from developmentally and regionally distinct subpopulations of neurons from the mouse hippocampus and broader nervous system. Multi-omics factor analyses revealed differing contributions of each transcriptomic layer in the discrimination of neurons based on their stage of development, region, and function. Importantly, profiles of differential alternative splicing and polyadenylation better discriminated specific neuronal subtype populations than gene expression patterns. These results provide evidence for differential relative contributions of coordinated gene regulatory layers in the specification of neuronal subtypes.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Microexons: at the nexus of nervous system development, behaviour and autism spectrum disorder.

The discovery and characterization of a network of highly conserved neuronal microexons has provided fundamental new insight into mechanisms underlying nervous system development and function, as well as an important basis for pathway convergence in autism spectrum disorder. In the past few years, considerable progress has been made in comprehensively determining the repertoires of factors that control neuronal microexons. These results have illuminated molecular mechanisms that activate the splicing of microexons, including those that control gene expression programs critical for neurogenesis, as well as synaptic protein translation and neuronal activity. Remarkably, individual disruption of specific microexons in these pathways results in autism-like phenotypes and cognitive impairment in mice. This review discusses these findings and their implications for delivering new therapeutic strategies for neurological disorders.

A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum.

Tight coordination of gene expression in the developing cerebellum is crucial for establishment of neuronal circuits governing motor and cognitive function. However, transcriptional changes alone do not explain all of the switches underlying neuronal differentiation. Here we unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. The motifs enriched in modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints by directly binding near the 3' splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, resulting in motor coordination defects, ataxia, and abnormal social behavior. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and establishes motor and cognitive circuitry in the developing cerebellum.

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.