A viral microRNA functions as an orthologue of cellular miR-155 - PubMed (original) (raw)
A viral microRNA functions as an orthologue of cellular miR-155
Eva Gottwein et al. Nature. 2007.
Abstract
All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.
Figures
Fig. 1. Expression of miR-K12−11 and miR-155 in BJAB cells
a. Alignment of mature miR-K12−11 and miR-155. b. Schematic of the lentiviral miRNA expression vector. c. Primer extension (PE) analysis of miRNA expression in BJAB transductants and control cell lines BC-1 and Jijoye for miR-K12−11, miR-155, miR-30/GL2, and cellular miR-16. d. Null distribution of miR-K12−11/ miR-155 seed abundance in 10,000 randomly sampled sets of 150 3’ UTRs, each from the space of our analysis. The arrow marks the abundance of 7mer seed matches in the 3’UTRs of the top-scoring 150 down-regulated genes in miR-K12−11 transductants, indicating a significant (p ≤ 0.0057) enrichment of potential targets for miR-K12−11/miR-155. Analysis for 6mer seed matches also identified a significant enrichment (p ≤ 0.001) (not shown). e. Mutations introduced to create miR-K12−11/2M are highlighted.
Fig. 2. miR-K12−11 and miR-155 regulate a common set of mRNAs
Real time qRT-PCR analysis of total RNA derived from two independent replicates of BJAB transductants, expressing no miRNA, miR-K12−11, miR-K12−11/2M, miR-155 or miR-30/GL2, for six candidate mRNA targets of miR-K12−11 and miR155. Relative RNA abundance is shown as a percentage of the level of 18S rRNA and error bars are derived from quadruplicate 18S rRNA replicates. mRNAs tested included BACH1 (a), FOS (b), BIRC4BP (c), AGTRAP (d), SAMHD1 (e), and RFK (f). Results for further candidate targets are shown in Suppl. Fig. 3.
Fig. 3. Direct and equivalent regulation of candidate 3’UTRs by miR-K12−11 and miR-155
Indicator vectors carrying no additional sequences, or candidate 3’UTR sequences inserted 3’ to FLuc, were cotransfected with an internal RLuc control vector and either empty pNL-SIN-CMV-AcGFP (black bars), pNL-SIN-CMV-AcGFP expressing miR-K12−11 (blue), miR-K12−11/2M (grey) or miR-155 (yellow). Dual luciferase assays were carried out 48 hours later. For each pNL-SIN-CMV-AcGFP construct, Fluc to Rluc ratios were first normalized to the value obtained for the empty indicator vector and then to the activities obtained with pNL-SIN-CMV-AcGFP, which were set at 100%. BACH1, IKBKE, MAP3K10, SLA and RPS6KA3 are predicted targets for miR-155 (Suppl. Table 2).
Fig. 4. Endogenous BACH1 and FOS proteins are repressed by both miR-K12−11 and miR-155
a. Western analysis of BACH1 protein expression in independently generated BJAB transductants expressing no miRNA, miR-K12−11, miR-K12−11/2M, miR-155 or miR-30/GL2. b. Western analysis of BACH1 protein expression in THP-1 transductants. c. Western analysis of FOS protein. Replicate BJAB transductants expressing no miRNA, miR-K12−11, miR-K12−11/2M, miR-155 or miR-30/GL2 were serum starved for 26 hours and then treated with serum free RPMI or TPA for two hours. d. FOS expression in KSHV-infected cells is rescued by a miR-K12−11-specific antagomir. BCBL-1 cells were serum starved for 25 hours in the presence of 1 μM antagomir against miR-K12−1 or miR-K12−11 and then incubated in serum-free medium, or serum-free medium ± 20 ng/ml TPA for one hour.
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