Abstract
We have used deep sequencing to analyze the pattern of viral microRNA (miRNA) expression observed in the B-cell line BC-3, which is latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV). We recovered 14.6 x 10(6) total miRNA cDNA reads, of which a remarkable 92% were of KSHV origin. We detected 11 KSHV miRNAs as well as all 11 predicted miRNA or passenger strands from the miRNA duplex intermediate. One previously reported KSHV miRNA, miR-K9, was found to be mutationally inactivated. This analysis revealed that the 5' ends of 10 of the 11 KSHV miRNAs were essentially invariant, with significantly more variation being observed at the 3' end, a result which is consistent with the proposal that the 5'-proximal region of miRNAs is critical for target mRNA recognition. However, one KSHV miRNA, miR-K10-3p, was detected in two isoforms differing by 1 nucleotide (nt) at the 5' end that were present at comparable levels, and these two related KSHV miRNAs are therefore likely to target at least partially distinct mRNA populations. Finally, we also report the first detection of miRNA offset RNAs (moRs) in vertebrate somatic cells. moRs, which derive from primary miRNA (pri-miRNA) sequences that immediately flank the mature miRNA and miRNA strands, were identified flanking one or both sides of nine of the KSHV miRNAs. These data provide new insights into the pattern of miRNA processing in mammalian cells and indicate that this process is highly conserved during animal evolution.
Highlights
We have used deep sequencing to analyze the pattern of viral microRNA expression observed in the B-cell line BC-3, which is latently infected with Kaposi’s sarcoma-associated herpesvirus (KSHV)
One previously reported KSHV miRNA, miR-K9, was found to be mutationally inactivated. This analysis revealed that the 5 ends of 10 of the 11 KSHV miRNAs were essentially invariant, with significantly more variation being observed at the 3 end, a result which is consistent with the proposal that the 5-proximal region of miRNAs is critical for target mRNA recognition
We have performed deep sequencing of small RNAs expressed in the latently KSHV-infected primary effusion lymphoma (PEL) B-cell line BC-3 in order to more fully define the exact miRNA coding potential of KSHV and to ask whether an in-depth analysis of viral miRNA expression, which is expected to be more homogeneous than the cellular miRNA expression pattern, might reveal insights into miRNA processing in vertebrate somatic cells
Summary
We have used deep sequencing to analyze the pattern of viral microRNA (miRNA) expression observed in the B-cell line BC-3, which is latently infected with Kaposi’s sarcoma-associated herpesvirus (KSHV). MoRs, which derive from primary miRNA (pri-miRNA) sequences that immediately flank the mature miRNA and miRNA* strands, were identified flanking one or both sides of nine of the KSHV miRNAs. we report the first detection of miRNA offset RNAs (moRs) in vertebrate somatic cells. After export to the cytoplasm, the pre-miRNA is bound by a second RNase III enzyme, called Dicer, which cleaves the pre-miRNA ϳ22 bp from the base, leaving a second 2-nt 3Ј overhang, to generate the miRNA duplex intermediate [16] One strand of this duplex intermediate—the mature miRNA strand—is incorporated into the RNA-induced silencing complex (RISC) [14], while the second strand—referred to as the passenger or miRNA* strand—is degraded. MiR-K1 to miR-K11, were initially detected in studies that used conventional sequencing to analyze small RNAs expressed in latently KSHV-infected B cells, while a 12th KSHV miRNA, miR-K12, was predicted computationally and confirmed by Northern analysis [13]
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