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RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression, and dysfunctional RBPs underlie many human diseases. Proteome-wide discovery efforts predict thousands of novel RBPs, many of which lack canonical RNA-binding domains. Here, we present a hybrid ensemble RBP classifier (HydRA) that leverages information from both intermolecular protein interactions and internal protein sequence patterns to predict RNA-binding capacity with unparalleled specificity and sensitivity using support vector machine, convolutional neural networks and transformer-based protein language models. HydRA enables Occlusion Mapping to robustly detect known RNA-binding domains and to predict hundreds of uncharacterized RNA-binding domains. Enhanced CLIP validation for a diverse collection of RBP candidates reveals genome-wide targets and confirms RNA-binding activity for HydRA-predicted domains. The HydRA computational framework accelerates construction of a comprehensive RBP catalogue and expands the set of known RNA-binding protein domains.HighlightsHydRA combines protein-protein interaction and amino acid sequence information to predict RNA binding activity for 1,487 candidate genes.HydRA predicts RNA binding with higher specificity and sensitivity than current approaches, notably for RBPs without well-defined RNA-binding domains.Occlusion Mapping with HydRA enables RNA-binding domain discovery.Enhanced CLIP confirms HydRA RBP predictions with RNA-binding domain resolution.

SummaryMost human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such “binding-first” assays affect protein function, nonetheless, often remains unclear. Here, we describe a “function-first” proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/β. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/β. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The cross-linked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so cross-linking events specifically occur between proteins and pre-replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and cross-linked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ~7 days and VIR-CLASP takes 1 day.

SUMMARY Upon pathogen detection, the innate immune system triggers signaling events leading to upregulation of pro-inflammatory and anti-microbial mRNA transcripts. RNA-binding proteins (RBPs) interact with these critical mRNAs and regulate their fates at the post-transcriptional level. One such RBP is ELAVL1. Although significant progress has been made in understanding how embryonic lethal vision-like protein 1 (ELAVL1) regulates mRNAs, its target repertoire and binding distribution within an immunological context remain poorly understood. We overlap four high-throughput approaches to define its context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 transitions from binding overwhelmingly intronic sites to 3′ UTR sites upon immune stimulation of cells, binding previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate pathways essential to pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to understand their post-transcriptional regulatory roles within specific biological circuitries., In brief Rothamel et al. show that upon immune activation, the RNA-binding protein ELAVL1 accumulates in the cytoplasm and redistributes from introns to mRNA 3′ UTRs. 3′ UTR binding confers enrichment and transcript stability. Many top-ranking transcripts are interferon-stimulated genes (ISGs), indicating that ELAVL1 is a positive regulator of an innate immune response., Graphical Abstract

Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical “interferon signature genes” that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry., Pfizer Inc.

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses.

Objective: Circadian rhythms influence the timing of behavior, neurological diseases, and even death. Rare mutations in homologs of evolutionarily conserved clock genes are found in select pedigrees with extreme sleep timing, and there is suggestive evidence that certain common polymorphisms may be associated with self-reported day/night preference. However, no common polymorphism has been associated with the timing of directly observed human behavioral rhythms or other physiological markers of circadian timing at the population level. Methods: We performed a candidate gene association study with replication, evaluating associations between polymorphisms in homologs of evolutionarily conserved clock genes and the timing of behavioral rhythms measured by actigraphy. For validated polymorphisms, we evaluated associations with transcript expression and time of death in additional cohorts. Results: rs7221412, a common polymorphism near period homolog 1 (PER1), was associated with the timing of activity rhythms in both the discovery and replication cohorts (joint p ¼ 2.1 � 10 � 7 ). Mean activity timing was delayed by 67 minutes in rs7221412 GG versus rs7221412 AA homozygotes. rs7221412 also showed a suggestive time-dependent relationship with both cerebral cortex (p ¼ 0.05) and CD14 þ CD16 � monocyte (p ¼ 0.02) PER1 expression and an interesting association with time of death (p ¼ 0.015) in which rs7221412 GG individuals had a mean time of death nearly 7 hours later than rs7221412 AA/AG . Interpretation: A common polymorphism near PER1 is associated with the timing of human behavioral rhythms, and shows evidence of association with time of death. This may be mediated by differential PER1 expression. These results may facilitate individualized scheduling of shift work, medical treatments, or monitoring of vulnerable patient populations. ANN NEUROL 2012;72:324–334

The previously published version of this Article contained errors in Fig. 6. In panel h the units of the x axis were incorrectly given as mM and should have been given as µM. Also, the IC50s for RU.365, RU.332 and RU.521 within panel h were incorrectly given as mM and should have been given as µM. These errors have been corrected in both the PDF and HTML versions of the Article.

Mostafavi, Sara et al. “Variation and Genetic Control of Gene Expression in Primary Immunocytes across Inbred Mouse Strains.” The Journal of Immunology 193.9 (2014): 4485–4496., National Institutes of Health (U.S.) (Immunological Genome Project. R24 AI072073)

As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory model provide a basis for additional hypothesis-based research on the importance of changes in gene expression in neutrophils in different conditions.

Much of the knowledge about cell differentiation and function in the immune system has come from studies in mice, but the relevance to human immunology, diseases, and therapy has been challenged, perhaps more from anecdotal than comprehensive evidence. To this end, we compare two large compendia of transcriptional profiles of human and mouse immune cell types. Global transcription profiles are conserved between corresponding cell lineages. The expression patterns of most orthologous genes are conserved, particularly for lineage-specific genes. However, several hundred genes show clearly divergent expression across the examined cell lineages, and among them, 169 genes did so even with highly stringent criteria. Finally, regulatory mechanisms—reflected by regulators’ differential expression or enriched cis -elements—are conserved between the species but to a lower degree, suggesting that distinct regulation may underlie some of the conserved transcriptional responses.

The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4[superscript +]CD8[superscript +] stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4[superscript +] or CD8[superscript +] lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation., National Institutes of Health (U.S.) (AI072073)