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ABSTRACT Direct RNA nanopore sequencing allows for the identification of full-length RNAs with a ∼10% error rate consisting of mismatches and small deletions. These errors are thought to be randomly distributed and structure-independent since RNA/cDNA duplexes are generated to prevent RNA structure formation prior to sequencing. When analyzing citrus yellow vein associated virus (CY1) reads during infection ofNicotiana benthamiana,viral (+/-)foldback RNAs (i.e., viral plus [+]-strands joined to [-]-strands) showed significantly higher error rates (mismatches and deletions) in the 5ʹ (+)RNA portion with errors that were relatively evenly distributed, while errors in the attached (-)RNA portion were less frequent and unevenly distributed. Non-foldback CY1 (+)RNAs from infected plants also showed an uneven distribution of errors, which correlated with errors inin vitrotranscribed CY1 (+)RNA reads in both position and frequency. Hotspot errors in non-foldback CY1 (+)RNA and (-)RNA reads only weakly correlated, and hotspots were frequently located 5ʹ of known structural elements. Since nanopore sequencing is also used to identify RNA modifications, which depend on base-specific sequencing errors, algorithms for RNA modification detection were also examined for bias. We found that multiple programs predicted RNA modifications inin vitrotranscribed CY1 RNA at the same positions and with similar confidence levels as within plantaCY1 RNA. These data suggest that direct RNA sequencing contains inherent error biases that may be associated with post-translocation RNA folding and low sequence complexity, and therefore extrapolations based on sequencing error require special consideration. 

ABSTRACT Virus-induced gene silencing (VIGS) allows for the rapid targeting of gene expression and has been instrumental in characterizing plant genes. However, foreign sequences inserted into VIGS vectors are rarely maintained for unknown reasons. Citrus yellow vein-associated umbravirus-like virus (CY1) with its solved secondary structure was converted into a VIGS vector to determine why simple hairpins inserted into non-functional, single-stranded locations are not maintained. When CY1 contained foreign hairpins with thermodynamic properties (positional entropy and/or ΔG) differing from those of natural CY1 hairpins, deletions arose within a few weeks of infectingNicotiana benthamiana. In contrast, duplication and insertion of four natural CY1 hairpins (up to 200 nt) into the same locations were retained until plant senescence. Hairpins containing similar conformations and thermodynamic properties as natural hairpins were also retained, as were hairpins that shared thermodynamic properties but were conformationally distinct. By predicting and modulating these thermodynamic properties, a hairpin was retained by CY1 for at least 30 months in citrus. These findings strongly suggest that RNA viruses have evolved to contain substructures with specific thermodynamic properties, and hairpins containing these properties are stable when inserted into non-functional regions of the genome, opening up VIGS for long-lived trees and vines. IMPORTANCEPlus-strand RNA plant viruses are used as tools to introduce small interfering RNAs (siRNAs) into laboratory plants to target and silence genes. However, virus-induced gene silencing (VIGS) vectors engineered to contain foreign hairpins or other sequences for siRNA generation are not stable, and the foreign sequences are rapidly lost. We found that foreign sequences are not maintained in an umbravirus-like VIGS vector (CY1) because their physical properties conflict with the innate properties of the CY1 genome’s substructures (i.e., hairpins). When natural CY1 hairpins were duplicated and inserted into locations where previous inserts were rapidly lost, the hairpins were now stable as were unrelated hairpins with the same physical properties. By mimicking the physical properties of the viral genome, one insert was stable for over 30 months. These results suggest that RNA viral genomes have evolved to have specific physical properties, and these properties appear to be similar for other plus-strand RNA viruses. 

Two-dimensional drawing of nucleic acid structures, particularly RNA structures, is fundamental to the communication of nucleic acids research. However, manually drawing structures is laborious and infeasible for structures thousands of nucleotides long. RNAcanvas automatically arranges residues into strictly shaped stems and loops while providing robust interactive editing features, including click-and-drag layout adjustment. Drawn elements are highly customizable in a point-and-click manner, including colours, fonts, size and shading, flexible numbering, and outlining of bases. Tertiary interactions can be drawn as draggable, curved lines. Leontis-Westhof notation for depicting non-canonical base-pairs is fully supported, as well as text labels for structural features (e.g., hairpins). RNAcanvas also has many unique features and performance optimizations for large structures that cannot be correctly predicted and require manual refinement based on the researcher’s own analyses and expertise. To this end, RNAcanvas has point-and-click structure editing with real-time highlighting of complementary sequences and motif search functionality, novel features that greatly aid in the identification of putative long-range tertiary interactions, de novo analysis of local structures, and phylogenetic comparisons. For ease in producing publication quality figures, drawings can be exported in both SVG and PowerPoint formats. URL: https://rnacanvas.app 

The 3′ untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3′UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3′ cap-independent translation enhancers (3′CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3′TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3′ terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed “Trio”, that are exclusively found in a subset of umbraviruses and are located just upstream of the 3′TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3′ terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3′CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement. 

Abstract RNA secondary (2D) structure visualization is an essential tool for understanding RNA function. R2DT is a software package designed to visualize RNA 2D structures in consistent, recognizable, and reproducible layouts. The latest release, R2DT 2.0, introduces multiple significant features, including the ability to display position-specific information, such as single nucleotide polymorphisms or SHAPE reactivities. It also offers a new template-free mode allowing visualization of RNAs without pre-existing templates, alongside a constrained folding mode and support for animated visualizations. Users can interactively modify R2DT diagrams, either manually or using natural language prompts, to generate new templates or create publication-quality images. Additionally, R2DT features faster performance, an expanded template library, and a growing collection of compatible tools and utilities. Already integrated into multiple biological databases, R2DT has evolved into a comprehensive platform for RNA 2D visualization, accessible at https://r2dt.bio. 

Abstract Canonical eukaryotic mRNA translation requires 5′cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5′cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3′UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson–Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson–Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions. 

ABSTRACT The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3′ untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3′ UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana , p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3′ UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor. IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3′ untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.