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Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation

ABSTRACT: The influenza polymerase cleaves host RNAs ~10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs.

ORGANISM(S): Influenza A virus (A/WSN/1933(H1N1))

PROVIDER: GSE65101 | GEO | 2015/04/23

SECONDARY ACCESSION(S): PRJNA272999

REPOSITORIES: GEO

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Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation

Project description:The influenza polymerase cleaves host RNAs ~10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs. Sixteen datasets were generated, corresponding to template-switching 5’-RACE libraries of 1) each viral RNA, 2) a time-course of NS1 viral RNA, or 3) CIP-TAP libraries of NS1 and PB2. Libraries contain random barcodes of length 5 (NS1_TS, PB2_TS, NS1_5R, PB2_5R) or 9 (other datasets) at the start of each read, and at least three guanosine residues for the template-switching datasets. See methods of accompanying paper for further details. Samples were sequenced on an Illumina HiSeq 2000.

2015-04-23 | E-GEOD-65101 | biostudies-arrayexpress

Influenza A virus preferentially snatches non-coding RNA caps

Project description:Influenza A virus (IAV) lacks the enzyme for adding 5â caps to its RNAs, and thus snatches the 5â ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched, nor the effect of âcap-snatchingâ on host processes has been completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNA from infected cells or relied solely on annotated host protein-coding genes to define host mRNAs selected by the virus. To examine the substrate-product relationship between all host RNAs, including non-coding RNAs, and viral RNAs, we used an unbiased approach to identify the host and viral capped RNAs from IAV-infected cells. We demonstrate that IAV predominantly snatches caps from non-coding host RNAs, particularly U1 and U2 small nuclear RNAs (snRNAs). Because snRNAs regulate host mRNA processing, cap-snatching of snRNAs may constitute a means by which IAV hijacks host cell metabolism. examine caps snatched by influenza virus A

2015-11-23 | E-GEOD-67493 | biostudies-arrayexpress

Influenza A virus preferentially snatches non-coding RNA caps

Project description:Influenza A virus (IAV) lacks the enzyme for adding 5’ caps to its RNAs, and thus snatches the 5’ ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched, nor the effect of “cap-snatching” on host processes has been completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNA from infected cells or relied solely on annotated host protein-coding genes to define host mRNAs selected by the virus. To examine the substrate-product relationship between all host RNAs, including non-coding RNAs, and viral RNAs, we used an unbiased approach to identify the host and viral capped RNAs from IAV-infected cells. We demonstrate that IAV predominantly snatches caps from non-coding host RNAs, particularly U1 and U2 small nuclear RNAs (snRNAs). Because snRNAs regulate host mRNA processing, cap-snatching of snRNAs may constitute a means by which IAV hijacks host cell metabolism.

2015-11-23 | GSE67493 | GEO

RNA-dependent RNA polymerase sequence specificities of capsnatching viruses are tailored to aid viral replication

Project description:Influenza A virus (IAV) is a major respiratory pathogen that causes severe illness and mortality worldwide. IAV critically depends on host factors for multiple functions that are not encoded by the viral genome. Notably, the IAV RNA-dependent RNA polymerase (RdRP) complex lacks intrinsic 7-methylguanosine (m7G) capping activity and instead “snatches” the 5’m7G caps and the adjacent 10-15nts of host transcripts to prime viral mRNA production, allowing viral transcripts to be recognized by the host translation machinery. We developed a new approach to simultaneously assay the 5’ ends of host and viral transcripts in infected cells and identify the host capsnatch origins for viral mRNAs. We used this technique to study the progression of IAV infection in nuclear and cytoplasmic compartiments and determined that the RdRP complex has specific sequence preferences that results in preferential capsnatching and avoidance of sub-populations of host RNAs, which can be reverted by the addition of a cap-snatch inhibitor. Notably, genes avoided by CapSnatch Seq have biological functions enriched for mRNA processing, translation, and viral replication. This selectivity and functional enrichment of avoided genes is conserved among capsnatching viruses including Influenza B Virus and Lymphocytic Choriomeningitis Virus, despite differences in RdRP domains and subcellular locations of RNA substrates. This suggests that this preference convergently arose as a viral strategy to allow cap-snatching viruses to avoid targeting genes necessary for its own replication.

2017-08-09 | GSE85474 | GEO

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains

Project description:Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)- containing domain translocates ~90 A ̊ to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.</br></br>Extra contact information:</br><a href="mailto:cusack@embl.fr">Stephen Cusack</a>, EMBL Grenoble Outstation, Unit of Virus Host-Cell Interactions, France ( corresponding author and lab head )

2016-01-04 | PXD002234 | Pride

HUMAN-VIRUS CHIMERIC PROTEINS GENERATED DURING INFECTION

Project description:RNA viruses are a major threat to global human health. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, as viral polymerases cleave 5′m7G-capped host transcripts to prime viral mRNA synthesis (‘cap-snatching’). We hypothesized that start codons within cap-snatched host transcripts could drive the expression of chimeric human-viral coding sequences. Here, we report the existence of this mechanism of gene origination (‘start-snatching’), which creates, depending on the translatability of the viral UTRs, human-virus protein chimeras either as N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, can generate T cell responses and contribute to virulence. Our results indicate that IAV, and likely a multitude of other human-, animal- and plant-viruses, use this host-dependent mechanism to expand their proteome diversity during infection.

2020-05-15 | GSE148245 | GEO

Nudt12 is an mRNA DeNADding Enzyme

Project description:The 5´-end 7-methylguanosine cap structure has long been known as a signature feature of eukaryotic cellular and viral mRNAs that confers mRNA stability and efficient translation. Recent findings in diverse organisms have demonstrated that RNAs can additionally possess a non-canonical cap structure consisting of a nicotinamide adenosine dinucleotide (NAD+) at their 5´ end in place of m7G. It has been shown that 5´ end-NAD+ cap promotes rapid decay of the RNA at least in part by the DXO family of proteins in mammalian cells. This observation led to the hypothesis that mammalian cells harbor additional deNADding enzymes that may function in distinct pathways. Here we report Nudt12 efficiently removes NAD+ caps and functions as alternate cellular deNADding enzyme that targets NAD+-capped RNAs distinct from DXO. Importantly, with the use of an NAD-Cap Detection (NAD-CapD) approach that utilizes enzymatic properties to release intact NAD+/NADH from the 5´ end of NAD-capped cellular RNAs and a colorimetric NAD Quantitation to detect released NAD+/NADH, we can follow total cellular NAD+ cap levels. Removal of Nudt12 or DXO deNADding enzymes from cells significantly increased levels of NAD+-capped cellular RNAs. Moreover, fungal Rai1 and Dxo1, previously demonstrated to possess deNADding activity in vitro, can also function as deNADding enzymes in yeast cells. Double disruption of Rai1 and Dxo1 in yeast cells lead to accumulation of NAD+-capped RNAs, indicating that both enzymes function to clear NAD+ from the 5´ end of RNAs. Finally, our findings established that alterations in cellular NAD+ levels impact NAD+-capped RNA levels implying NAD+ capping is a modulated process that may be linked to the metabolic state of the cell.

2019-05-20 | GSE110801 | GEO

The RNA NAD+ cap is widespread in the Arabidopsis transcriptome and probably promotes translation

Project description:As the most common mRNA cap, the m7G cap impacts the fate of an mRNA in eukaryotes. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD+), can be used as an initiating nucleotide in RNA synthesis to result in NAD+-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD+ cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD+- capped RNAs are widespread in Arabidopsis thaliana. NAD+-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes but not the chloroplast genome. NAD-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD+-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms of translation initiation on NAD+-capped mRNAs. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, and maybe regulated by, mRNA NAD capping.

2019-06-03 | GSE127002 | GEO

Respiratory Syncytial Virus (RSV) optimizes the translational landscape during infection

Project description:Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5’-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5’-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation.

2024-09-03 | GSE268742 | GEO

Landscape of RNA NAD+ capping in Arabidopsis unopened flower bud

Project description:Eukaryotic messenger RNAs (mRNAs) possess a 5’-end N7-methyl guanosine (m7G) cap that promotes their translation and stability. However, it was recently demonstrated that eukaryotic mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD+) cap that promotes mRNA decay mediated by the NAD+ decapping enzyme DXO1. However, the dynamic regulation of NAD+ capping in plant remains unknown. Here, we describe the global landscape of NAD+-capped RNAs in Arabidopsis thaliana, and demonstrate that DXO1 is responsible for removal of these 5’-end modifications and facilitates mRNA degradation in plant transcriptomes. We also reveal that in the absence of DXO1 NAD+-capped mRNAs are unstable and processed into smRNAs. Furthermore, we find that Abscisic Acid (ABA) remodel the landscape of RNA cap epitransciptome, and the mRNA lost their NAD+ cap contribute to their stability under ABA. Overall, our results support a link between ABA response and RNA NAD+ capping.

2020-11-23 | GSE142389 | GEO

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