Novel validated transcripts. Bottom track contains novel EBV transcripts validated in this study. B Annotation status of validated transcripts. C Number of GenBank-annotated transcripts validated in this study. Novel intergenic transcripts. Gray shaded track displays GenBank-annotated features. Error bars are standard deviation. Programmed exon skipping in the W repeat region.
Readthrough transcription and intergenic splicing at the BZLF2 locus. Black arrows indicate transcripts whose largest ORF is an in-frame fusion. Despite previous findings of pervasive transcription of the EBV genome outside of GenBank-annotated genes 9 , 12 , 30 , 35 , 36 , the structures of transcripts derived from these regions could not be directly ascertained. To gain further insights into the functional nature of BCLT transcripts we examined their coding potential and subcellular location.
BHRF1 transcripts containing W1 and W2 exons have been detected previously in latently infected cells 41 — 43 so the omission of the W2 exon observed here may be unique to the setting of reactivation. Together these results indicate that there is a unique and specific alternative splicing program across the W repeats during reactivation.
Interestingly, while two different open reading frames extend across the repeated W1 exons, we did not detect any AUG start codons corresponding to either of these reading frames. Microarray studies previously showed that the latency-associated LMP2 gene is induced during reactivation in type I latency cells 29 , More recent RNA-Seq work from our lab revealed extensive alternative splicing of LMP2 transcripts during reactivation that is not observed in type III latency LMP2 transcripts 12 , possibly representing a means of increasing the functional diversity of this locus during reactivation.
This supports the idea that some lytic LMP2-overlapping isoforms play non-coding nuclear functions while others are transported to the cytoplasm for translation. Together, these findings illustrate the complex nature of the lytic LMP2 transcriptome arising from alternative promoter usage, alternative splicing and differential cellular localization, producing a diverse group of LMP2-overlapping transcripts likely with both coding and non-coding functions.
While we observed poly A signal readthrough transcription throughout the genome including at the LMP2 locus , transcripts originating from the BZLF2 promoter BZLF2p were remarkable with respect to the diversity of transcripts initiating from a single promoter including coding, non-coding and chimeric transcripts. Two transcripts initiating at this promoter contain the entire BZLF2 open reading frame encoding the membrane glycoprotein gp42 and terminate about 2 kilobases downstream from the translation stop codon at a pair of novel polyadenylation sites located 23 bases apart.
These transcripts likely represent the 2. Notably, both transcripts are potentially bicistronic, containing a second, larger downstream open reading frame that we previously reported based on RNA-Seq data 9. The differential use of polyadenylation signals combined with extensive alternative splicing results in over 30 different validated transcript structures. While the entire locus of BZLF2p-derived transcripts spans more than 19 kb, reverse transcriptase processivity limitations may have precluded the detection of additional longer BZLF2 isoforms.
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Further, we have fully resolved transcripts that had previously only been observed as splice junctions or cDNA fragments 9 , 12 , 31 , TRIMD analysis of the cellular transcriptome also reveals an abundance of previously unannotated isoforms unpublished , including previously unannotated splice variants and alterations of transcript ends. Since cellular isoform expression is known to be regulated across different tissues and conditions 44 — 46 TRIMD-based transcript structure resolution may have an important application in globally resolving tissue, developmental and disease specific cellular transcript structures.
Based on this analysis, we estimate that there may be more than polyadenylated EBV transcripts expressed during reactivation Supplementary Figure S6. Even this number may under-represent the full complexity of the EBV lytic transcriptome because it only accounts for polyadenylated transcripts. A hallmark of viruses is their compact nature, with an uncanny density of functions encoded in relatively small amounts of genomic sequence. The work presented here helps highlight some of the many strategies utilized by EBV to achieve diverse functions with limited genetic material.
Coincident with the finding of novel lytic splice junctions, we observed extensive alternative splicing e. The extent to which the lytic restriction of alternative splicing is controlled by unique trans factors versus cis -acting regulatory elements is unclear. While the functions of these newly discovered alternatively spliced lytic transcripts are still unknown, it is likely that alternative splicing specifies distinct functions.
Some alternative splicing occurs within coding sequences, giving rise to transcripts encoding proteins with different repertoires of functional domains. This may represent a conservation strategy involving the multi-purposing of functional domains to accomplish distinct tasks in the viral infection cycle.
The expression of several latency genes during reactivation has been shown previously to occur through distinct lytic promoters e. The use of distinct promoters may be a means of facilitating cleaner transcriptional regulation through the segregation of latency- and lytic-specific promoter elements. As mentioned above, this seems to be the case with LMP2 transcripts initiating from different lytic promoters, some of which appear to play non-coding roles in the nucleus while others are exported to the cytoplasm for translation.
The use of multiple promoters to drive expression of common downstream sequences is not unique to the LMP2 locus or latency genes in general but is common among purely lytic genes as well. For example, transcripts containing BZLF1 sequences are derived from previously known and novel upstream promoter elements Supplementary Figure S7.
We also note downstream promoter usage e. Alternative promoter usage is observed across the genome and likely contributes to the generation of substantial diversity in transcript function. The readthrough of polyadenylation signals generates a substantial amount of transcript diversity during EBV reactivation e.
Interestingly, a recent report showed that HSV-1 infection results in massive transcriptional readthrough in the cellular genome Although the functional significance of readthrough transcription of the cellular genome is unclear at this time, a global induction of polyadenylation site readthrough mechanisms may be essential for the generation of transcript diversity during the EBV replication cascade. Beyond the functional relevance of the growing number of EBV transcripts being identified, pervasive overlapping lytic transcription has practical implications for experimental design.
Investigations into viral gene function through genome modification need to take into account the full repertoire of transcripts potentially impacted by the introduced genetic alterations. Further, even antisense directed knockdown strategies can have unintended influences on overlapping transcripts.
It is clear that investigations into EBV gene function require continued efforts to fully resolve the viral transcriptome as well as careful experimental design and interpretation of results. The complex nature of the EBV lytic transcriptome also has implications for lytic transcript quantification.clublavoute.ca/xogaf-donde-conocer-gente.php
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Accurately quantifying the abundance of coexpressed cellular gene isoforms using short-read sequencing data has been a challenge, although significant progress has been made 17 , At a minimum, it will be important to fully resolve the EBV transcriptome to better facilitate transcript quantification. Simulations can then be performed to benchmark the accuracy of viral transcript quantification using the most complete transcript structure information.
Nevertheless, we may find that lytic transcript quantification can only be achieved through the implementation of novel strategies using long-read data or a combination of platforms similar to the approaches used here for transcript resolution. National Center for Biotechnology Information , U. Journal List Nucleic Acids Res v. Nucleic Acids Res. Published online Jul Erik K. Author information Article notes Copyright and License information Disclaimer. For commercial re-use, please contact moc. This article has been cited by other articles in PMC.
Abstract Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Determination of full-length coverage percentages of expressed cellular genes All human RNA transcripts with RefSeq Reviewed or Validated status were evaluated. Naming of new transcripts The naming scheme for novel transcripts is based on the existing naming scheme for EBV genes.
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Calculation of coding potential Sequences of validated isoforms were analyzed for coding potential and the presence of open reading frames with the Coding Potential Assessment Tool Open in a separate window. Figure 1. Limitations in attributing bona fide transcript structures from Iso-Seq data Examination of CFLs that mapped to well-characterized cellular genes revealed those that agree with RefSeq annotation as well as those that appear to reflect novel variants. Transcriptome resolution through integration of multi-platform data TRIMD While certain assumptions could potentially be made to impute ambiguously presenting CFL structural features e.
Figure 6. Figure 3. Figure 4. Figure 5. Figure 7. Novel intergenic transcripts Despite previous findings of pervasive transcription of the EBV genome outside of GenBank-annotated genes 9 , 12 , 30 , 35 , 36 , the structures of transcripts derived from these regions could not be directly ascertained. Complex lytic promoter usage for LMP2 transcripts Microarray studies previously showed that the latency-associated LMP2 gene is induced during reactivation in type I latency cells 29 , Intergenic splicing of BZLF2 transcripts arising from readthrough transcription While we observed poly A signal readthrough transcription throughout the genome including at the LMP2 locus , transcripts originating from the BZLF2 promoter BZLF2p were remarkable with respect to the diversity of transcripts initiating from a single promoter including coding, non-coding and chimeric transcripts.
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