Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site.

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, FIONA1, 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.

All the information necessary to build the proteins that perform the biological processes required for life is encoded in the DNA of an organism. Making these proteins requires the DNA sequence of a gene to be transcribed into a 'messenger RNA' (mRNA), which is then processed into a final, mature form. This blueprint is then translated to assemble the corresponding protein. When an mRNA is processed, segments of the sequence that do not code for protein are removed and the remaining coding sequences are joined together in the right order. An intricate molecular machine known as the spliceosome controls this mechanism by recognising the 'splice sites' where coding and non-coding sequences meet. Depending on external conditions, the spliceosome can 'pick-and-mix' the coding sequences to create different processed mRNAs (and therefore proteins) from a single gene. This alternative splicing mechanism is often used to regulate when certain biological processes take place based on environmental cues; for example, the splicing of genes which control the timing of plant flowering is sensitive to ambient temperatures. To investigate this mechanism, Parker et al. focused on Arabidopsis thaliana, a plant that blooms later when temperatures are low. This precise timing partly relies on a gene whose mRNA is efficiently spliced in the cold, resulting in an active form of its protein that blocks blooming. Parker et al. grew and screened many A. thaliana plants to find individuals that could flower early in the cold, in which splicing of this gene was disrupted. A mutant fitting these criteria was identified and subjected to further investigation, which revealed that it could not produce FIONA1. In non-mutant plants, this enzyme chemically modifies one of the components of the spliceosome, a small nuclear RNA known as U6. Parker et al found that there are two types of splice site ­ one more likely to interact with U6 and another that preferentially interacts with another small nuclear RNA, U5. When FIONA1 is inactive (such as in the mutant identified by Parker et al.), splice sites that tend to strongly interact with U5 are selected. However, when the enzyme is active, splice sites that tend to bind with the chemically modified U6 are used instead. Further work by Parker et al. showed that these two types of splice sites ('preferring' either U5 or U6) are found in equal proportions in the genomes of many species, including humans. This suggests that Parker et al. have uncovered an essential feature of how genomes are organised and splicing is controlled.

Chromosome evolution and the genetic basis of agronomically important traits in greater yam.

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.

Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA.

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.

Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification.

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.

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch.

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 Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNAIle 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 tRNAIle and the other between the Stem IIA/B pseudoknot and the D loop of tRNAIle 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.

Riboswitch-Mediated Gene Regulation: Novel RNA Architectures Dictate Gene Expression Responses.

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.

T box riboswitches in Actinobacteria: translational regulation via novel tRNA interactions.

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 tRNA-dependent 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.