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RNA. 2010 Aug;16(8):1540-58. doi: 10.1261/rna.1967910. Epub 2010 Jun 21.

Small RNA profiling reveals antisense transcription throughout the KSHV genome and novel small RNAs.

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1
Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX 78712-0162, USA.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus that encodes 12 precursor microRNAs (pre-miRNAs) that give rise to 17 different known approximately 22-nucleotide (nt) effector miRNAs. Like all herpesviruses, KSHV has two modes of infection: (1) a latent mode whereby only a subset of viral genes are expressed and (2) a lytic mode during which the full remaining viral genes are expressed. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. Here, we developed a method to profile small RNAs ( approximately 18-75 nt) from populations of cells undergoing predominantly lytic infection. Using two different next-generation sequencing platforms, we cloned and sequenced both pre-miRNAs and derivative miRNAs. Our analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5' end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. We report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miR-K12-8 and pre-miR-K12-12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. We identify several novel small RNAs of low abundance, including viral miRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. Finally, we observe widespread antisense transcription relative to known coding sequences during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, we observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs).

PMID:
20566670
PMCID:
PMC2905754
DOI:
10.1261/rna.1967910
[Indexed for MEDLINE]
Free PMC Article
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