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Direct RNA sequencing of adenovirus

ABSTRACT: Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

PROVIDER: PRJEB35652 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	ERR3772610_1.fastq.gz	Fastqsanger.gz
	ERR3772610_2.fastq.gz	Fastqsanger.gz
	ERR3772601_1.fastq.gz	Fastqsanger.gz
	ERR3772601_2.fastq.gz	Fastqsanger.gz

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N6-methyladenosine modification is not a general trait of positive-sense RNA viral genomes

Project description:<p>Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m6A in CHIKV and DENV RNAs. For this, we combined m6A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m6A machinery’s localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.</p>

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Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Project description:Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORF), six novel ORF-containing transcripts, and fifteen transcripts encoding for messages that potentially alter protein functions through truncations or fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Of these, an evolutionary conserved protein was detected containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.

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Primary neonatal mouse cardiomyocytes: empty adenovirus infection vs. Mettl14-overexpression adenovirus infection

Project description:The adenovirus carrying Mettl14 gene infects primary neonatal mouse cardiomyocytes, which is in contrast with the primary neonatal mouse cardiomyocytes infected with empty virus. The purpose is to explore the effect of Mettl14 on the global mRNA m6A modification level in primary neonatal rat cardiomyocytes.

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Project description:Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants that lack the virus-directed ubiquitin ligase required for efficient processing of viral RNA. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively promote efficient splicing and mRNA processing to limit dsRNA formation, thus avoiding detection and restriction by host nucleic acid sensors.

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Project description:We report the direct RNA sequencing of HEK293 and a primary human mammary epithelial cell (HMEC) line using Oxford Nanopore based sequencing. Using this data, we built an algorithm to detect m6A modifications within the DRACH motif context. Evaluation of m6A sites was carried out with HEK METTL3 knockdown and HMEC ALKBH5 over expression cell lines.

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