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RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells. N.M. was supported by an FPI fellowship from the Ministry of Economy and Competitiveness; F.G. was supported by national grants BFU2015-68741-P from MINECO, PGC2018-099697-B-I00 from MICINN, ‘la Caixa’ Foundation [100010434] under the agreement LCF/PR/HR17/52150016, Marató-TV3 Foundation [20131430]; Catalan Agency for Research and Universities [2017SGR534]; CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is member of ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I + D + i 2013–2016 from the Instituto de Salud Carlos III (ISCIII), ERDF, and ‘Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya’ [2017SGR595]. A.C. is funded by the MRC Career Development Award (MR/L019434/1) and the ERC Consolidator 101001634 vRNP-capture. We acknowledge the support of the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI /10.13039/501100011033), and the Generalitat de Catalunya through the CERCA Programme. Funding for open access charge: MINECO [PGC2018-099697-B-I00].

SummaryWhile post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation.Brief outlineA novel high resolution and sensitive approach to systematically co-visualise the distribution of mRNAs and proteins in the intact nervous system reveals that post-transcriptional regulation of gene expression is very common. The rich data landscape is provided as a browsable resource (link), using Zegami, a cloud-based data exploration platform (link). Our solution provides a paradigm for the characterisation of post-transcriptional regulation of most genes and model systems.Highlights196/200 (98%) Drosophila genes show discordant RNA and protein expression in at least one nervous system region137/200 (68.5%) mRNAs are present in at least one synaptic compartmentNovel localised mRNA and protein discovered in periphery of glial processesNew paradigm for analysis of post-transcriptional regulation and data exploration

SummaryDespite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.Graphical AbstractIn briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain.HighlightsSingle molecule quantification of SARS-CoV-2 replication uncovers early infection kineticsThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replicationGenomic RNA is stable and persistent during the initial stages of infectionB.1.1.7 variant replicates more slowly than the Victoria strain

The expansion of tropical mosquito habitats and associated arboviruses is a risk for human health, and it thus becomes fundamental to identify new antiviral strategies. In this study we employ a new approach to elucidate the composition of the ribonucleoproteins (RNPs) of a prototypical arbovirus called Sindbis (SINV). SINV RNPs contain 453 cellular and 6 viral proteins, many of these proteins are nuclear in uninfected cells and redistribute to the cytoplasm upon infection. These findings suggest that SINV RNAs act as ’spiderwebs’, capturing host factors required for viral replication and gene expression in the cytoplasm. Functional perturbation of several of these host proteins causes profound effects in virus infection, as illustrated here with the tRNA ligase complex. Moreover, inhibition of viral RNP components with available drugs hampers the infection of a wide range of viruses, opening new avenues for the development of broad-spectrum therapies.Research highlightsSINV RNA interactome includes 453 cellular and 6 viral proteins.Nuclear RBPs that interact with SINV RNA are selectively redistributed to the cytoplasm upon infectionThe tRNA ligase complex plays major regulatory roles in SINV and SARS-CoV- 2 replicationThe SINV RNA interactome is enriched in pan-viral regulators with therapeutic potential.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19., Graphical Abstract, Kamel, Noerenberg, Cerikan and colleagues apply a multi-omic approach to identify the RNA-binding proteins that regulate SARS-CoV-2 infection. They discovered that the complement of RNA-binding proteins heavily remodels upon SARS-CoV-2 infection. They also show that the viral RNA interacts with dozens of cellular and six viral RNA-binding proteins. These host-virus interactions are fundamental for SARS-CoV-2 infection and have great potential for new therapeutic approaches against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

Highlights • Proteomic studies have uncovered more than a thousand cellular proteins that are present in the particles of RNA viruses. • Strikingly, nearly four hundred of these are RNA-binding proteins (RBPs). • Several virion-incorporated RBPs have been shown to regulate viral particle assembly and the initial steps of infection. • These host RBPs with regulatory roles in infection represent potential targets for antiviral therapy., RNA is a central molecule in RNA virus biology due to its dual function as messenger and genome. However, the small number of proteins encoded by viral genomes is insufficient to enable virus infection. Hence, viruses hijack cellular RNA-binding proteins (RBPs) to aid replication and spread. In this review we discuss the ‘knowns’ and ‘unknowns’ regarding the contribution of host RBPs to the formation of viral particles and the initial steps of infection in the newly infected cell. Through comparison of the virion proteomes of ten different human RNA viruses, we confirm that a pool of cellular RBPs are typically incorporated into viral particles. We describe here illustrative examples supporting the important functions of these RBPs in viral particle formation and infectivity and we propose that the role of host RBPs in these steps can be broader than previously anticipated. Understanding how cellular RBPs regulate virus infection can lead to the discovery of novel therapeutic targets against viruses.

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates., eLife digest The brain is a highly complex organ made up of huge numbers of different cell types that connect up to form a precise network. All these different cell types are generated from the repeated division of a relatively small pool of cells called neural stem cells. The division of these cells needs to be carefully regulated so that the correct number and type of nerve cells are produced at the right time and place. But it remains unclear how the division rate of individual neural stem cells is controlled during development. Controlling these divisions requires the activity of countless genes to be tightly regulated over space and time. When a gene is active, it is copied via a process called transcription into a single-stranded molecule known as messenger RNA (or mRNA for short). This molecule provides the instructions needed to build the protein encoded within the gene. Proteins are the functional building blocks of all cells. The conventional way of controlling protein levels is to vary the number of mRNA molecules made by transcription. Now, Samuels et al. reveal a second mechanism of determining protein levels in the brain, through regulating the stability of mRNA after it is transcribed. Samuels et al. discovered that a key regulatory protein called Imp controls the growth and division of individual neural stem cells in the brains of developing fruit flies. The experiments showed that Imp binds to mRNA molecules that contain the code for a protein called Myc, which is known to drive cell growth and division in many different cell types. Both human Imp and Myc have been implicated in cancer. Using a technique that images single molecules of mRNA, Samuels et al. showed that the Imp protein in stem cells stabilises the mRNA molecule coding for Myc. This means that when more Imp is present, more Myc protein gets produced. Thus, the level of Imp in each individual neural stem cell fine-tunes the rate at which the cell grows and divides: the higher the level of Imp, the larger the stem cell and the faster it divides. These findings underscore how important post-transcriptional processes are for regulating gene activity in the developing brain. The methods used in this study to study mRNA molecules in single cells also provide new insights that could not be derived from the average measurements of many cells. Similar methods could also be applied to other developmental systems in the future.

The human immunodeficiency virus type 1 (HIV-1) proteome is expressed from alternatively spliced and unspliced genomic RNAs. However, HIV-1 RNAs that are not fully spliced are perceived by the host machinery as defective and are retained in the nucleus. During late infection, HIV-1 bypasses this regulatory mechanism by expression of the Rev protein from a fully spliced mRNA. Once imported into the nucleus, Rev mediates the export of unprocessed HIV-1 RNAs to the cytoplasm, leading to the production of the viral progeny. While regarded as a canonical RNA export factor, Rev has also been linked to HIV-1 RNA translation, stabilization, splicing and packaging. However, Rev's functions beyond RNA export have remained poorly understood. Here, we revisit this paradigmatic protein, reviewing recent data investigating its structure and function. We conclude by asking: what remains unknown about this enigmatic viral protein?

The crucial participation of cellular RNA‐binding proteins (RBPs) in virtually all steps of virus infection has been known for decades. However, most of the studies characterizing this phenomenon have focused on well‐established RBPs harboring classical RNA‐binding domains (RBDs). Recent proteome‐wide approaches have greatly expanded the census of RBPs, discovering hundreds of proteins that interact with RNA through unconventional RBDs. These domains include protein–protein interaction platforms, enzymatic cores, and intrinsically disordered regions. Here, we compared the experimentally determined census of RBPs to gene ontology terms and literature, finding that 472 proteins have previous links with viruses. We discuss what these proteins are and what their roles in infection might be. We also review some of the pioneering examples of unorthodox RBPs whose RNA‐binding activity has been shown to be critical for virus infection. Finally, we highlight the potential of these proteins for host‐based therapies against viruses. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional ImplicationsRNA in Disease and Development > RNA in DiseaseRNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes, The compendium of RNA‐binding proteins related to virus infection reveals an unexplored world of unconventional virus–host interactions.

Cancer development involves the stepwise accumulation of genetic lesions that overcome the normal regulatory pathways that prevent unconstrained cell division and tissue growth. Identification of the genetic changes that cause cancer has long been the subject of intensive study, leading to the identification of several RNA-binding proteins (RBPs) linked to cancer. Cross-reference of the complement of RBPs recently identified by RNA interactome capture with cancer-associated genes and biological processes led to the identification of a set of 411 proteins with potential implications in cancer biology. These involve a broad spectrum of cellular processes including response to stress, metabolism and cell adhesion. Future studies should aim to understand these proteins and their connection to cancer from an RNA-centred perspective, holding the promise of new mechanistic understanding of cancer formation and novel approaches to diagnosis and treatment.

Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here, we find that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but due to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provides a framework with which evolution shapes transcriptomes.

Cell-to-cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3' untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3' isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome-wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single-cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single-cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3' isoform choice has potential implications on functional cell-to-cell heterogeneity as well as utility in resolving cell populations.

Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non‐coding transcriptional events from each genic locus using a novel genome‐wide, nucleotide‐resolution technique to quantify the half‐lives of 3′ transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3′ untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3′ UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA‐protein interactions in conditioning isoform‐specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.

Alternative polyadenylation site usage gives rise to variation in 3′ ends of transcripts in diverse organisms ranging from yeast to human. Accurate mapping of polyadenylation sites of transcripts is of major biological importance, since the length of the 3′UTR can have a strong influence on transcript stability, localization, and translation. However, reads generated using total mRNA sequencing mostly lack the very 3′ end of transcripts. Here, we present a method that allows simultaneous analysis of alternative 3′ ends and transcriptome dynamics at high throughput. By using transcripts produced in vitro , the high precision of end mapping during the protocol can be controlled. This method is illustrated here for budding yeast. However, this method can be applied to any natural or artificially polyadenylated RNA.

The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3 0 T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3 0 -untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3 0 -UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.