Direct identification of A-to-I editing sites with nanopore native RNA sequencing.

Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.

In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation.

Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach, sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), that maps pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra- and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes, including the modular organization of mRNAs, its impact on translation and decay, and the enrichment of long-range interactions in noncoding RNAs. Additionally, intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites, expanding our knowledge of rRNA biogenesis. These results highlight the underexplored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology.

Comprehensive mapping of SARS-CoV-2 interactions in vivo reveals functional virus-host interactions.

SARS-CoV-2 is a major threat to global health. Here, we investigate the RNA structure and RNA-RNA interactions of wildtype (WT) and a mutant (Δ382) SARS-CoV-2 in cells using Illumina and Nanopore platforms. We identify twelve potentially functional structural elements within the SARS-CoV-2 genome, observe that subgenomic RNAs can form different structures, and that WT and Δ382 virus genomes fold differently. Proximity ligation sequencing identify hundreds of RNA-RNA interactions within the virus genome and between the virus and host RNAs. SARS-CoV-2 genome binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2'-O-methylated. 2'-O-methylation sites are enriched in viral untranslated regions, associated with increased virus pair-wise interactions, and are decreased in host mRNAs upon virus infection, suggesting that the virus sequesters methylation machinery from host RNAs towards its genome. These studies deepen our understanding of the molecular and cellular basis of SARS-CoV-2 pathogenicity and provide a platform for targeted therapy.

Determination of isoform-specific RNA structure with nanopore long reads.

Current methods for determining RNA structure with short-read sequencing cannot capture most differences between distinct transcript isoforms. Here we present RNA structure analysis using nanopore sequencing (PORE-cupine), which combines structure probing using chemical modifications with direct long-read RNA sequencing and machine learning to detect secondary structures in cellular RNAs. PORE-cupine also captures global structural features, such as RNA-binding-protein binding sites and reactivity differences at single-nucleotide variants. We show that shared sequences in different transcript isoforms of the same gene can fold into different structures, highlighting the importance of long-read sequencing for obtaining phase information. We also demonstrate that structural differences between transcript isoforms of the same gene lead to differences in translation efficiency. By revealing isoform-specific RNA structure, PORE-cupine will deepen understanding of the role of structures in controlling gene regulation.

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.