Abstract
We have used high-throughput small RNA sequencing to characterize viral small RNA expression in purified tonsillar B and T lymphocytes isolated from patients tested positive for Epstein-Barr virus (EBV) or human adenovirus (HAdV) infections, respectively. In the small set of patients analyzed, the expression profile of EBV and HAdV miRNAs could not distinguish between patients diagnosed with tonsillar hypertrophy or chronic/recurrent tonsillitis. The EBV miR-BART expression profile among the patients diagnosed with tonsillar diseases resembles most closely the pattern seen in EBV+ tumors (Latency II/I). The miR-BARTs that appear to be absent in normal EBV infected cells are essentially all detectable in the diseased tonsillar B lymphocytes. In the EBV+ B cells we detected 44 EBV miR-BARTs derived from the proposed BART precursor hairpins whereof five are not annotated in miRBase v21. One previously undetected miRNA, BART16b-5p, originates from the miR-BART16 precursor hairpin as an alternative 5´ miR-BART16 located precisely upstream of the annotated miR-BART16-5p. Further, our analysis revealed an extensive sequence variation among the EBV miRNAs with isomiRs having a constant 5´ end but alternative 3´ ends. A range of small RNAs was also detected from the terminal stem of the EBER RNAs and the 3´ part of v-snoRNA1. During a lytic HAdV infection in established cell lines the terminal stem of the viral non-coding VA RNAs are processed to highly abundant viral miRNAs (mivaRNAs). In contrast, mivaRNA expression in HAdV positive tonsillar T lymphocytes was very low. The small RNA profile further showed that the 5´ mivaRNA from VA RNAI and the 3´ mivaRNA from VA RNAII were as predicted, whereas the 3´ mivaRNA from VA RNAI showed an aberrant processing upstream of the expected Dicer cleavage site.
Highlights
MicroRNAs are a large family of ~22-nucleotide noncoding RNAs expressed in multicellular eukaryotes and encoded by some viruses [1]
In a recent report we characterized the prevalence of Epstein-Barr virus (EBV) and human adenovirus (HAdV) infections in tonsillar B and T lymphocytes isolated from patients diagnosed with tonsillar diseases [25]
Since miRNAs have been implicated in a variety of diseases, we decided to define the viral small RNA profiles in the tonsillar B and T lymphocytes from the patients positive for EBV (EBV+) or HAdV (HAdV+) infections using high-throughput small RNA sequencing
Summary
MicroRNAs (miRNAs) are a large family of ~22-nucleotide (nt) noncoding RNAs expressed in multicellular eukaryotes and encoded by some viruses [1]. More than 2500 mature miRNA species have so far been reported [2]. In the canonical miRNA biogenesis pathway, the miRNA genes are transcribed by RNA polymerase II into a long primary miRNA transcript that is processed by the nuclear Drosha/ DGCR8 microprocessor complex, generating a 60–90 nt precursor miRNA (pre-miRNA) hairpin. After being exported to the cytoplasm the pre-miRNA is further processed by the endonuclease Dicer to yield a ~22-nt double-stranded miRNA composed of so-called 5p and 3p strands. One strand of the mature miRNA duplex (the guide strand) is loaded onto one of four Argonaute proteins (Ago) forming the so-called RNA-induced silencing complex (RISC) [3]. The guide strand directs the RISC complex to the target mRNAs for subsequent post-transcriptional gene silencing [1]
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