SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Exploring the expanding universe of host-virus interactions mediated by viral RNA.

RNA is a central molecule in RNA virus biology; however, the interactions that it establishes with the host cell are only starting to be elucidated. In recent years, a methodology revolution has dramatically expanded the scope of host-virus interactions involving the viral RNA (vRNA). A second wave of method development has enabled the precise study of these protein-vRNA interactions in a life cycle stage-dependent manner, as well as providing insights into the interactome of specific vRNA species. This review discusses these technical advances and describes the new regulatory mechanisms that have been identified through their use. Among these, we discuss the importance of vRNA in regulating protein function through a process known as riboregulation. We envision that the elucidation of vRNA interactomes will open new avenues of research, including pathways to the discovery of host factors with therapeutic potential against viruses.

Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle.

mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.

Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20.

Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and nonphosphorylated forms of mRBPs. Almost 200 phosphorylation events affected pull-down efficiency compared with the unmodified mRBPs and thus have regulatory potential. Our data capture known regulatory phosphorylation sites in ELAVL1, SF3B1, and UPF1 and identify potential regulatory sites. Follow-up experiments on the splicing regulator RBM20 revealed multiple phosphorylation sites in the C-terminal disordered region affecting nucleocytoplasmic localization, association with cytoplasmic ribonucleoprotein granules, and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.

The SARS-CoV-2 RNA interactome.

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

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.

High-throughput quantitation of protein-RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins.

UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions, however, makes attribution of RNA-binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA-binding protein (RBP) confidence scoring metric (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high- and low-confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions, for example, the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support the development of more predictive computational approaches for RBP identification.

A Hitchhiker's guide to RNA-RNA structure and interaction prediction tools.

RNA biology has risen to prominence after a remarkable discovery of diverse functions of noncoding RNA (ncRNA). Most untranslated transcripts often exert their regulatory functions into RNA-RNA complexes via base pairing with complementary sequences in other RNAs. An interplay between RNAs is essential, as it possesses various functional roles in human cells, including genetic translation, RNA splicing, editing, ribosomal RNA maturation, RNA degradation and the regulation of metabolic pathways/riboswitches. Moreover, the pervasive transcription of the human genome allows for the discovery of novel genomic functions via RNA interactome investigation. The advancement of experimental procedures has resulted in an explosion of documented data, necessitating the development of efficient and precise computational tools and algorithms. This review provides an extensive update on RNA-RNA interaction (RRI) analysis via thermodynamic- and comparative-based RNA secondary structure prediction (RSP) and RNA-RNA interaction prediction (RIP) tools and their general functions. We also highlighted the current knowledge of RRIs and the limitations of RNA interactome mapping via experimental data. Then, the gap between RSP and RIP, the importance of RNA homologues, the relationship between pseudoknots, and RNA folding thermodynamics are discussed. It is hoped that these emerging prediction tools will deepen the understanding of RNA-associated interactions in human diseases and hasten treatment processes.

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.

BACKGROUND: The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. RESULTS: Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. CONCLUSION: We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Viral RNA Is a Hub for Critical Host-Virus Interactions.

RNA is a central molecule in the life cycle of viruses, acting not only as messenger (m)RNA but also as a genome. Given these critical roles, it is not surprising that viral RNA is a hub for host-virus interactions. However, the interactome of viral RNAs remains largely unknown. This chapter discusses the importance of cellular RNA-binding proteins in virus infection and the emergent approaches developed to uncover and characterise them.

Comprehensive Profiling of Roquin Binding Preferences for RNA Stem-Loops.

The cellular levels of mRNAs are controlled post-transcriptionally by cis-regulatory elements located in the 3'-untranslated region. These linear or structured elements are recognized by RNA-binding proteins (RBPs) to modulate mRNA stability. The Roquin-1 and -2 proteins specifically recognize RNA stem-loop motifs, the trinucleotide loop-containing constitutive decay elements (CDEs) and the hexanucleotide loop-containing alternative decay elements (ADEs), with their unique ROQ domain to initiate mRNA degradation. However, the RNA-binding capacity of Roquin towards different classes of stem-loops has not been rigorously characterized, leaving its exact binding preferences unclear. Here, we map the RNA-binding preference of the ROQ domain at nucleotide resolution introducing sRBNS (structured RNA Bind-n-Seq), a customized RBNS workflow with pre-structured RNA libraries. We found a clear preference of Roquin towards specific loop sizes and extended the consensus motifs for CDEs and ADEs. The newly identified motifs are recognized with nanomolar affinity through the canonical RNA-ROQ interface. Using these new stem-loop variants as blueprints, we predicted novel Roquin target mRNAs and verified the expanded target space in cells. The study demonstrates the power of high-throughput assays including RNA structure formation for the systematic investigation of (structural) RNA-binding preferences to comprehensively identify mRNA targets and elucidate the biological function of RBPs.

RNA Conformation Capture by Proximity Ligation.

RNA proximity ligation is a set of molecular biology techniques used to analyze the conformations and spatial proximity of RNA molecules within cells. A typical experiment starts with cross-linking of a biological sample using UV light or psoralen, followed by partial fragmentation of RNA, RNA-RNA ligation, library preparation, and high-throughput sequencing. In the past decade, proximity ligation has been used to study structures of individual RNAs, networks of interactions between small RNAs and their targets, and whole RNA-RNA interactomes, in models ranging from bacteria to animal tissues and whole animals. Here, we provide an overview of the field, highlight the main findings, review the recent experimental and computational developments, and provide troubleshooting advice for new users. In the final section, we draw parallels between DNA and RNA proximity ligation and speculate on possible future research directions.

RBPome identification in egg-cell like callus of Arabidopsis.

RNA binding proteins (RBPs) have multiple and essential roles in transcriptional and posttranscriptional regulation of gene expression in all living organisms. Their biochemical identification in the proteome of a given cell or tissue requires significant protein amounts, which limits studies in rare and highly specialized cells. As a consequence, we know almost nothing about the role(s) of RBPs in reproductive processes such as egg cell development, fertilization and early embryogenesis in flowering plants. To systematically identify the RBPome of egg cells in the model plant Arabidopsis, we performed RNA interactome capture (RIC) experiments using the egg cell-like RKD2-callus and were able to identify 728 proteins associated with poly(A+)-RNA. Transcripts for 97 % of identified proteins could be verified in the egg cell transcriptome. 46 % of identified proteins can be associated with the RNA life cycle. Proteins involved in mRNA binding, RNA processing and metabolism are highly enriched. Compared with the few available RBPome datasets of vegetative plant tissues, we identified 475 egg cell-enriched RBPs, which will now serve as a resource to study RBP function(s) during egg cell development, fertilization and early embryogenesis. First candidates were already identified showing an egg cell-specific expression pattern in ovules.

Circulating microRNA trafficking and regulation: computational principles and practice.

Rapid advances in genomics discovery tools and a growing realization of microRNA's implication in intercellular communication have led to a proliferation of studies of circulating microRNA sorting and regulation across cells and different species. Although sometimes, reaching controversial scientific discoveries and conclusions, these studies have yielded new insights in the functional roles of circulating microRNA and a plethora of analytical methods and tools. Here, we consider this body of work in light of key computational principles underpinning discovery of circulating microRNAs in terms of their sorting and targeting, with the goal of providing practical guidance for applications that is focused on the design and analysis of circulating microRNAs and their context-dependent regulation. We survey a broad range of informatics methods and tools that are available to the researcher, discuss their key features, applications and various unsolved problems and close this review with prospects and broader implication of this field.

The expanding world of metabolic enzymes moonlighting as RNA binding proteins.

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme-RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme-RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme-RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme-RNA interactions in connecting intermediary metabolism and gene expression.

The increasing diversity and complexity of the RNA-binding protein repertoire in plants.

Post-transcriptional regulation has far-reaching implications on the fate of RNAs. It is gaining increasing momentum as a critical component in adjusting global cellular transcript levels during development and in response to environmental stresses. In this process, RNA-binding proteins (RBPs) are indispensable chaperones that naturally bind RNA via one or multiple globular RNA-binding domains (RBDs) changing the function or fate of the bound RNAs. Despite the technical challenges faced in plants in large-scale studies, several hundreds of these RBPs have been discovered and elucidated globally over the past few years. Recent discoveries have more than doubled the number of proteins implicated in RNA interaction, including identification of RBPs lacking classical RBDs. This review will discuss these new emerging classes of RBPs, focusing on the current state of the RBP repertoire in Arabidopsis thaliana, including the diverse functional roles derived from quantitative studies implicating RBPs in abiotic stress responses. Notably, this review highlights that 836 RBPs are enriched as Arabidopsis RBPs while 1865 can be classified as candidate RBPs. The review will also outline outstanding areas within this field that require addressing to advance our understanding and potential biotechnological applications of RBPs.

Functions and properties of nuclear lncRNAs-from systematically mapping the interactomes of lncRNAs.

Protein and DNA have been considered as the major components of chromatin. But beyond that, an increasing number of studies show that RNA occupies a large amount of chromatin and acts as a regulator of nuclear architecture. A significant fraction of long non-coding RNAs (lncRNAs) prefers to stay in the nucleus and cooperate with protein complexes to modulate epigenetic regulation, phase separation, compartment formation, and nuclear organization. An RNA strand also can invade into double-stranded DNA to form RNA:DNA hybrids (R-loops) in living cells, contributing to the regulation of gene expression and genomic instability. In this review, we discuss how nuclear lncRNAs orchestrate cellular processes through their interactions with proteins and DNA and summarize the recent genome-wide techniques to study the functions of lncRNAs by revealing their interactomes in vivo.

IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories.

RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurological disease. Developing axons provide a model well suited to the study of RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing mouse brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal that it is required for normal axon pathfinding during vertebrate development.

Specific RNP capture with antisense LNA/DNA mixmers.

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.