Search for: All records

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. 

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.