Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

Riboswitches are RNA elements that act on the mRNA with which they are cotranscribed to modulate expression of that mRNA. These elements are widely found in bacteria, where they have a broad impact on gene expression. The defining feature of riboswitches is that they directly recognize a physiological signal, and the resulting shift in RNA structure affects gene regulation. The majority of riboswitches respond to cellular metabolites, often in a feedback loop to repress synthesis of the enzymes used to produce the metabolite. Related elements respond to the aminoacylation status of a specific tRNA or to a physical parameter, such as temperature or pH. Recent studies have identified new classes of riboswitches and have revealed new insights into the molecular mechanisms of signal recognition and gene regulation. Application of structural and biophysical approaches has complemented previous genetic and biochemical studies, yielding new information about how different riboswitches operate.

The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNAdependent stabilization of a helix that sequesters a sequence complementary to the Shine-Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNA Ile and tRNA Ile -dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch.

The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an orphan crop. Here, we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using the genomic tools, we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here, we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIO1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing.

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNA ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNA and the other between the Stem IIA/B pseudoknot and the D loop of tRNA These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

21Understanding genome organization and gene regulation requires insight into RNA transcription, 22 processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a 23 wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA 24 methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide 25 and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, 26 poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3' untranslated regions is associated 27 with decreased relative transcript abundance and defective RNA 3′ end formation. A functional 28 consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore 29 direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length 30 single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying 31 this approach to less well-studied species could transform our understanding of what their genomes 32 encode. 33 34 misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events 46 produced by RT template switching 4,5 , and the inability to detect all base modifications in the 47 copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to 48 interpret the combination of authentic RNA processing events and remains an unsolved problem 7 . 49We investigated whether long-read direct RNA sequencing (DRS) with nanopores 8 could 50 reveal the complexity of Arabidopsis mRNA processing and modifications. In nanopore DRS, the 51 protein pore (nanopore) sits in a membrane through which an electrical current is passed, and intact 52 RNA is fed through the nanopore by a motor protein 8 . Each RNA sequence within the nanopore 53 (5 bases) can be identified by the magnitude of signal it produces. Arabidopsis is a pathfinder model 54 in plant biology, and its genome annotation strongly influences the annotation and our 55 understanding of what other plant genomes encode. We applied nanopore DRS and Illumina RNAseq 56 to wild-type Arabidopsis (Col-0) and mutants defective in m 6 A 9 and exosome-mediated RNA decay 10 . 57We reveal m 6 A and combinations of RNA processing events (alternative patterns of 5′ capped 58 transcription start sites, splicing, 3′ polyadenylation and poly(A) tail length) in full-length Arabidopsis 59 mRNAs transcriptome-wide. 60 61 Results 62Nanopore DRS detects long, complex mRNAs and short, structured non-coding RNAs 63We purified poly (A)+ RNA from four biological replicates of 14-day-old Arabidopsis Col-0 seedlings. 64We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all 65 replicates 11,12 and carried out nanopore DRS. Illumina RNAseq was performed in parallel on similar 66 material. Using Guppy base-calling (Oxford Nanopore Technologies) and minimap2 alignment 67 software 13 , we identified around 1 mi...