Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (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. Among them are 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.

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis.

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.

The importance of virion-incorporated cellular RNA-Binding Proteins in viral particle assembly and infectivity.

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.

Protein Syndesmos is a novel RNA-binding protein that regulates primary cilia formation.

Syndesmos (SDOS) is a functionally poorly characterized protein that directly interacts with p53 binding protein 1 (53BP1) and regulates its recruitment to chromatin. We show here that SDOS interacts with another important cancer-linked protein, the chaperone TRAP1, associates with actively translating polyribosomes and represses translation. Moreover, we demonstrate that SDOS directly binds RNA in living cells. Combining individual gene expression profiling, nucleotide crosslinking and immunoprecipitation (iCLIP), and ribosome profiling, we discover several crucial pathways regulated post-transcriptionally by SDOS. Among them, we identify a small subset of mRNAs responsible for the biogenesis of primary cilium that have been linked to developmental and degenerative diseases, known as ciliopathies, and cancer. We discover that SDOS binds and regulates the translation of several of these mRNAs, controlling cilia development.

Single-cell polyadenylation site mapping reveals 3' isoform choice variability.

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.

The new (dis)order in RNA regulation.

RNA-binding proteins play a key role in the regulation of all aspects of RNA metabolism, from the synthesis of RNA to its decay. Protein-RNA interactions have been thought to be mostly mediated by canonical RNA-binding domains that form stable secondary and tertiary structures. However, a number of pioneering studies over the past decades, together with recent proteome-wide data, have challenged this view, revealing surprising roles for intrinsically disordered protein regions in RNA binding. Here, we discuss how disordered protein regions can mediate protein-RNA interactions, conceptually grouping these regions into RS-rich, RG-rich, and other basic sequences, that can mediate both specific and non-specific interactions with RNA. Disordered regions can also influence RNA metabolism through protein aggregation and hydrogel formation. Importantly, protein-RNA interactions mediated by disordered regions can influence nearly all aspects of co- and post-transcriptional RNA processes and, consequently, their disruption can cause disease. Despite growing interest in disordered protein regions and their roles in RNA biology, their mechanisms of binding, regulation, and physiological consequences remain poorly understood. In the coming years, the study of these unorthodox interactions will yield important insights into RNA regulation in cellular homeostasis and disease.

Alternative polyadenylation diversifies post-transcriptional regulation by selective RNA-protein interactions.

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.

An efficient method for genome-wide polyadenylation site mapping and RNA quantification.

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'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'-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'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.

Functional consequences of bidirectional promoters.

Several studies have shown that promoters of protein-coding genes are origins of pervasive non-coding RNA transcription and can initiate transcription in both directions. However, only recently have researchers begun to elucidate the functional implications of this bidirectionality and non-coding RNA production. Increasing evidence indicates that non-coding transcription at promoters influences the expression of protein-coding genes, revealing a new layer of transcriptional regulation. This regulation acts at multiple levels, from modifying local chromatin to enabling regional signal spreading and more distal regulation. Moreover, the bidirectional activity of a promoter is regulated at multiple points during transcription, giving rise to diverse types of transcripts.

Systematic analysis of YFP traps reveals common mRNA/protein discordance in neural tissues.

While 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 lines across the intact Drosophila nervous system. 97.5% 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 visualization tools for post-transcriptional regulation.

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.

Despite 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, subgenomic 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.

Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability.

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.

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.

Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation.

During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the stability of the long prospero mRNA isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long prospero isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.This article has an associated First Person interview with the first author of the paper.

Unconventional RNA-binding proteins step into the virus-host battlefront.

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 Implications RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Expanding horizons: new roles for non-canonical RNA-binding proteins in cancer.

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.

Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap.

This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters.

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 found 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 owing 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, provide a framework with which evolution shapes transcriptomes.

Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality.

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3' ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Assessment of metagenomic assembly using simulated next generation sequencing data.

Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition. For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities. Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available.