Sequencing accuracy and systematic errors of nanopore direct RNA sequencing.

BACKGROUND: Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied. RESULTS: We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits. CONCLUSIONS: As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.

Recruitment of multi-segment genomic RNAs by Bluetongue virus requires a preformed RNA network.

How do segmented RNA viruses correctly recruit their genome has yet to be clarified. Bluetongue virus is a double-stranded RNA virus with 10 segments of different sizes, but it assembles its genome in single-stranded form through a series of specific RNA-RNA interactions prior to packaging. In this study, we determined the structure of each BTV transcript, individually and in different combinations, using 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP identified RNA structural changes during complex formation and putative RNA-RNA interaction sites. Our data also revealed a core RNA-complex of smaller segments which serves as the foundation ('anchor') for the assembly of a complete network composed of ten ssRNA segments. The same order of core RNA complex formation was identified in cells transfected with viral RNAs. No viral protein was required for these assembly reactions. Further, substitution mutations in the interacting bases within the core assemblies, altered subsequent segment addition and affected virus replication. These data identify a wholly RNA driven reaction that may offer novel opportunities for designed attenuation or antiviral therapeutics.

Isoform-specific RNA structure determination using Nano-DMS-MaP.

RNA structure determination is essential to understand how RNA carries out its diverse biological functions. In cells, RNA isoforms are readily expressed with partial variations within their sequences due, for example, to alternative splicing, heterogeneity in the transcription start site, RNA processing or differential termination/polyadenylation. Nanopore dimethyl sulfate mutational profiling (Nano-DMS-MaP) is a method for in situ isoform-specific RNA structure determination. Unlike similar methods that rely on short sequencing reads, Nano-DMS-MaP employs nanopore sequencing to resolve the structures of long and highly similar RNA molecules to reveal their previously hidden structural differences. This Protocol describes the development and applications of Nano-DMS-MaP and outlines the main considerations for designing and implementing a successful experiment: from bench to data analysis. In cell probing experiments can be carried out by an experienced molecular biologist in 3-4 d. Data analysis requires good knowledge of command line tools and Python scripts and requires a further 3-5 d.

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.

Advanced fluorescence microscopy in respiratory virus cell biology.

Respiratory viruses are a major public health burden across all age groups around the globe, and are associated with high morbidity and mortality rates. They can be transmitted by multiple routes, including physical contact or droplets and aerosols, resulting in efficient spreading within the human population. Investigations of the cell biology of virus replication are thus of utmost importance to gain a better understanding of virus-induced pathogenicity and the development of antiviral countermeasures. Light and fluorescence microscopy techniques have revolutionized investigations of the cell biology of virus infection by allowing the study of the localization and dynamics of viral or cellular components directly in infected cells. Advanced microscopy including high- and super-resolution microscopy techniques available today can visualize biological processes at the single-virus and even single-molecule level, thus opening a unique view on virus infection. We will highlight how fluorescence microscopy has supported investigations on virus cell biology by focusing on three major respiratory viruses: respiratory syncytial virus (RSV), Influenza A virus (IAV) and SARS-CoV-2. We will review our current knowledge of virus replication and highlight how fluorescence microscopy has helped to improve our state of understanding. We will start by introducing major imaging and labeling modalities and conclude the chapter with a perspective discussion on remaining challenges and potential opportunities.

Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging.

A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.

Nano-DMS-MaP allows isoform-specific RNA structure determination.

Genome-wide measurements of RNA structure can be obtained using reagents that react with unpaired bases, leading to adducts that can be identified by mutational profiling on next-generation sequencing machines. One drawback of these experiments is that short sequencing reads can rarely be mapped to specific transcript isoforms. Consequently, information is acquired as a population average in regions that are shared between transcripts, thus blurring the underlying structural landscape. Here, we present nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP)-a method that exploits long-read sequencing to provide isoform-resolved structural information of highly similar RNA molecules. We demonstrate the value of Nano-DMS-MaP by resolving the complex structural landscape of human immunodeficiency virus-1 transcripts in infected cells. We show that unspliced and spliced transcripts have distinct structures at the packaging site within the common 5' untranslated region, likely explaining why spliced viral RNAs are excluded from viral particles. Thus, Nano-DMS-MaP is a straightforward method to resolve biologically important transcript-specific RNA structures that were previously hidden in short-read ensemble analyses.

Cis-mediated interactions of the SARS-CoV-2 frameshift RNA alter its conformations and affect function.

The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through -1 programmed ribosomal frameshifting (PRF). -1PRF in the 1a/1b gene is essential for efficient viral replication and transcription of the viral genome. -1PRF efficiency relies on the presence of conserved RNA elements within the FSE. One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5' and 3' immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.

Short- and long-range interactions in the HIV-1 5' UTR regulate genome dimerization and packaging.

RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5' untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5' UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS-SL1 interaction unexpectedly couples the PBS with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.

Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55Gag and its maturation products.

Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting.

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

RNA Structures and Their Role in Selective Genome Packaging.

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

The evolution of RNA structural probing methods: From gels to next-generation sequencing.

RNA molecules are important players in all domains of life and the study of the relationship between their multiple flexible states and the associated biological roles has increased in recent years. For several decades, chemical and enzymatic structural probing experiments have been used to determine RNA structure. During this time, there has been a steady improvement in probing reagents and experimental methods, and today the structural biologist community has a large range of tools at its disposal to probe the secondary structure of RNAs in vitro and in cells. Early experiments used radioactive labeling and polyacrylamide gel electrophoresis as read-out methods. This was superseded by capillary electrophoresis, and more recently by next-generation sequencing. Today, powerful structural probing methods can characterize RNA structure on a genome-wide scale. In this review, we will provide an overview of RNA structural probing methodologies from a historical and technical perspective. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in vitro and In Silico RNA Methods > RNA Analyses in Cells.

RNA Structure-A Neglected Puppet Master for the Evolution of Virus and Host Immunity.

The central dogma of molecular biology describes the flow of genetic information from DNA to protein via an RNA intermediate. For many years, RNA has been considered simply as a messenger relaying information between DNA and proteins. Recent advances in next generation sequencing technology, bioinformatics, and non-coding RNA biology have highlighted the many important roles of RNA in virtually every biological process. Our understanding of RNA biology has been further enriched by a number of significant advances in probing RNA structures. It is now appreciated that many cellular and viral biological processes are highly dependent on specific RNA structures and/or sequences, and such reliance will undoubtedly impact on the evolution of both hosts and viruses. As a contribution to this special issue on host immunity and virus evolution, it is timely to consider how RNA sequences and structures could directly influence the co-evolution between hosts and viruses. In this manuscript, we begin by stating some of the basic principles of RNA structures, followed by describing some of the critical RNA structures in both viruses and hosts. More importantly, we highlight a number of available new tools to predict and to evaluate novel RNA structures, pointing out some of the limitations readers should be aware of in their own analyses.

Structural and Functional Motifs in Influenza Virus RNAs.

Influenza A viruses (IAV) are responsible for recurrent influenza epidemics and occasional devastating pandemics in humans and animals. They belong to the Orthomyxoviridae family and their genome consists of eight (-) sense viral RNA (vRNA) segments of different lengths coding for at least 11 viral proteins. A heterotrimeric polymerase complex is bound to the promoter consisting of the 13 5'-terminal and 12 3'-terminal nucleotides of each vRNA, while internal parts of the vRNAs are associated with multiple copies of the viral nucleoprotein (NP), thus forming ribonucleoproteins (vRNP). Transcription and replication of vRNAs result in viral mRNAs (vmRNAs) and complementary RNAs (cRNAs), respectively. Complementary RNAs are the exact positive copies of vRNAs; they also form ribonucleoproteins (cRNPs) and are intermediate templates in the vRNA amplification process. On the contrary, vmRNAs have a 5' cap snatched from cellular mRNAs and a 3' polyA tail, both gained by the viral polymerase complex. Hence, unlike vRNAs and cRNAs, vmRNAs do not have a terminal promoter able to recruit the viral polymerase. Furthermore, synthesis of at least two viral proteins requires vmRNA splicing. Except for extensive analysis of the viral promoter structure and function and a few, mostly bioinformatics, studies addressing the vRNA and vmRNA structure, structural studies of the influenza A vRNAs, cRNAs, and vmRNAs are still in their infancy. The recent crystal structures of the influenza polymerase heterotrimeric complex drastically improved our understanding of the replication and transcription processes. The vRNA structure has been mainly studied in vitro using RNA probing, but its structure has been very recently studied within native vRNPs using crosslinking and RNA probing coupled to next generation RNA sequencing. Concerning vmRNAs, most studies focused on the segment M and NS splice sites and several structures initially predicted by bioinformatics analysis have now been validated experimentally and their role in the viral life cycle demonstrated. This review aims to compile the structural motifs found in the different RNA classes (vRNA, cRNA, and vmRNA) of influenza viruses and their function in the viral replication cycle.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry.

The HIV-1 Pr55Gag precursor specifically selects genomic RNA (gRNA) from a large variety of cellular and spliced viral RNAs (svRNAs), however the molecular mechanisms of this selective recognition remains poorly understood. To gain better understanding of this process, we analyzed the interactions between Pr55Gag and a large panel of viral RNA (vRNA) fragments encompassing the main packaging signal (Psi) and its flanking regions by fluorescence spectroscopy. We showed that the gRNA harbors a high affinity binding site which is absent from svRNA species, suggesting that this site might be crucial for selecting the HIV-1 genome. Our stoichiometry analysis of protein/RNA complexes revealed that few copies of Pr55Gag specifically associate with the 5' region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55Gag binding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55Gag. Our analysis of gRNA fragments of different length supports the existence of a long-range tertiary interaction involving sequences upstream and downstream of the Psi region. This long-range interaction might promote optimal exposure of SL1 for efficient Pr55Gag recognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55Gag among a variety of svRNAs, all harboring SL1 in their first common exon.

The Life-Cycle of the HIV-1 Gag-RNA Complex.

Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55(Gag) precursor polyprotein, and the processes leading to its incorporation into viral particles.

MIMEAnTo: profiling functional RNA in mutational interference mapping experiments.

The mutational interference mapping experiment (MIME) is a powerful method that, coupled to a bioinformatics analysis pipeline, allows the identification of domains and structures in RNA that are important for its function. In MIME, target RNAs are randomly mutated, selected by function, physically separated and sequenced using next-generation sequencing (NGS). Quantitative effects of each mutation at each position in the RNA can be recovered with statistical certainty using the herein developed user-friendly, cross-platform software MIMEAnTo (MIME Analysis Tool). AVAILABILITY AND IMPLEMENTATION: MIMEAnTo is implemented in C ++ using the boost library as well as Qt for the graphical user interface and is distributed under GPL (http://www.gnu.org/licences/gpl). The libraries are statically linked in a stand alone executable and are not required on the system. The plots are generated with gnuplot. Gnuplot-iostream (https://github.com/dstahlke/gnuplot-iostream) serves as gnuplot interface. Standalone executables including examples and source code can be downloaded from https://github.com/maureensmith/MIMEAnTo CONTACTS: msmith@zedat.fu-berlin.de or vkleist@zedat.fu-berlin.deSupplementary information: Supplementary data are available at Bioinformatics online.