Dataset Information

Long-Read Isoform Sequencing Reveals a Hidden Complexity of the Transcriptional Landscape of Herpes Simplex Virus Type 1.

ABSTRACT: In this study, we used the amplified isoform sequencing technique from Pacific Biosciences to characterize the poly(A)+ fraction of the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). Our analysis detected 34 formerly unidentified protein-coding genes, 10 non-coding RNAs, as well as 17 polycistronic and complex transcripts. This work also led us to identify many transcript isoforms, including 13 splice and 68 transcript end variants, as well as several transcript overlaps. Additionally, we determined previously unascertained transcriptional start and polyadenylation sites. We analyzed the transcriptional activity from the complementary DNA strand in five convergent HSV gene pairs with quantitative RT-PCR and detected antisense RNAs in each gene. This part of the study revealed an inverse correlation between the expressions of convergent partners. Our work adds new insights for understanding the complexity of the pervasive transcriptional overlaps by suggesting that there is a crosstalk between adjacent and distal genes through interaction between their transcription apparatuses. We also identified transcripts overlapping the HSV replication origins, which may indicate an interplay between the transcription and replication machineries. The relative abundance of HSV-1 transcripts has also been established by using a novel method based on the calculation of sequencing reads for the analysis.

SUBMITTER: Tombacz D

PROVIDER: S-EPMC5476775 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Long-Read Isoform Sequencing Reveals a Hidden Complexity of the Transcriptional Landscape of Herpes Simplex Virus Type 1.

Tombácz Dóra D Csabai Zsolt Z Szűcs Attila A Balázs Zsolt Z Moldován Norbert N Sharon Donald D Snyder Michael M Boldogkői Zsolt Z

Frontiers in microbiology 20170620

In this study, we used the amplified isoform sequencing technique from Pacific Biosciences to characterize the poly(A)<sup>+</sup> fraction of the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). Our analysis detected 34 formerly unidentified protein-coding genes, 10 non-coding RNAs, as well as 17 polycistronic and complex transcripts. This work also led us to identify many transcript isoforms, including 13 splice and 68 transcript end variants, as well as several transcript overl ...[more]

PMID: 28676792

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Project description:Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying polycistronic RNAs, transcript isoforms including splice and transcript end variants, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 2,300 previously undetected transcripts, including coding, and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we found previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5' regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.

Project description:Even though alternative RNA splicing was discovered in 1977 (nearly 50 years ago), we still understand very little about most isoforms arising from a single gene, including in which tissues they are expressed and if their functions differ. Human gene annotations suggest remarkable transcriptional complexity, with approximately 252,798 distinct RNA isoform annotations from 62,710 gene bodies (Ensembl v109; 2023), emphasizing the need to understand their biological effects. For example, 256 gene bodies have ≥50 annotated isoforms and 30 have ≥100, where one protein-coding gene (MAPK10) even has 192 distinct RNA isoform annotations. Whether such isoform diversity results from biological noise (i.e., spurious alternative splicing) or whether it represents biological intent and specialized functions (even if subtle) remains a mystery. Recent studies by Aguzzoli-Heberle et al., Leung et al., and Glinos et al. demonstrate long-read RNAseq enables improved RNA isoform quantification for essentially any tissue, cell type, or biological condition (e.g., disease, development, aging, etc.) making it possible to better assess individual isoform expression and function. While each study provided important discoveries related to RNA isoform diversity, deeper exploration is needed. We sought, in part, to quantify real isoform usage across tissues (compared to annotations) and explore whether observed diversity is biological noise or intent. We used long-read RNAseq data from 58 GTEx samples across nine tissues (three brain, two heart, muscle, lung, liver, and cultured fibroblasts) generated by Glinos et al. and found considerable isoform diversity within and across tissues. Cerebellar hemisphere was the most transcriptionally complex tissue (22,522 distinct isoforms; 3,726 unique); liver was least diverse (12,435 isoforms; 1,039 unique). We highlight gene clusters exhibiting high tissue-specific isoform diversity per tissue (e.g., TPM1 expresses 19 in heart's atrial appendage), and specific genes (PAX6 and TPM1) that counterintuitively exhibit evidence that their expressed isoform diversity results from both biological noise and intent. We also validated 447 of the 700 new isoforms discovered by Aguzzoli-Heberle et al. and found that 88 were expressed in all nine tissues, while 58 were specific to a single tissue. This study represents a broad survey of the RNA isoform landscape, demonstrating isoform diversity across nine tissues and emphasizes the need to better understand how individual isoforms from a single gene body contribute to human health and disease.

Dataset Information

Long-Read Isoform Sequencing Reveals a Hidden Complexity of the Transcriptional Landscape of Herpes Simplex Virus Type 1.

Publications

Long-Read Isoform Sequencing Reveals a Hidden Complexity of the Transcriptional Landscape of Herpes Simplex Virus Type 1.

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