The miR-122 binding site in HCV displays position-dependent function on gene expression when located in the 5’ or 3’NCR of reporter mRNAs
The binding site for miR-122 in the HCV 5’UTR shows characteristics of a typical miRNA binding site. Specifically, HCV RNA nucleotides 23 to 28 display Watson-Crick base pair complementarity to the microRNA seed sequence (Lewis et al., 2005
), that encompasses nucleotides 2–7 (Jopling et al., 2005
). We have shown recently that the interaction of miR-122 with the viral genome is essential for the accumulation of viral RNA in cultured Huh7 liver cells (Jopling et al., 2005
To determine whether the upregulating function of the miR-122 binding site is dependent on the surrounding viral sequence context and its location at the 5’ end of the viral mRNA, we inserted nucleotides 1–347 of the HCV type 1a genome into the 5’NCR of a firefly luciferase reporter plasmid, designated pHCV-Luc (). The plasmid was introduced into Huh7 cells together with a control Renilla luciferase expressing plasmid, pRL-SV40. Luciferase activity was measured in the presence of co-transfected 2’O-ribose methylated miR-122 antisense oligonucleotides (miR-122 antisense) with exact complementarity to miR-122, which were shown to sequester intracellular miR-122 molecules and to inhibit their activity in the RNA interfering pathway (Jopling et al., 2005
). Randomized 2’O-methylated RNA oligonucleotides (Random-2’OMe), known to have no effect on miR-122 activity (Jopling et al., 2005
), served as negative controls. The data in show that the intracellular abundance of miR-122 had no effect on the efficiency of reporter mRNA expression. Similarly, reporter gene expression was not affected after ectopic expression of synthetic wild-type miR-122 molecules (miR-122 wt) (). Control Renilla luciferase activities were similar in each of these experiments (data not shown). These data argue that miR-122 binding sites do not affect the efficiency with which the HCV internal ribosome entry site directs protein synthesis when located at the 5’ end of reporter mRNAs.
Fig. 1 Expression of reporter mRNAs containing the HCV 5’ terminal sequences in their 5’ noncoding sequences. A: Diagram of capped Renilla luciferase-encoding reporter mRNA, expressed from plasmid pRL-SV40, and of firefly luciferase-encoding (more ...)
To examine whether miR-122 affects reporter gene expression from miR-122 binding sites in 3’NCRs, HCV sequences 1–45 were inserted into the 3’NCR of the firefly luciferase reporter gene to yield plasmid pLUC-122x1 (). Luciferase activity was measured in the presence of co-transfected Random-2’OMe oligonucleotides and set as 100 (). Addition of miR-122 antisense oligonucleotides enhanced firefly luciferase activity by approximately 30% (). In contrast, ectopic expression of synthetic miR-122 wt molecules () led to a 50% decrease in luciferase activity (). Ectopic expression of mutated miR-122p3-4 RNAs (), which should not bind to wildtype miR-122 seed match sequences (Jopling et al., 2005
), had no effect on reporter mRNA expression (). Thus, it appears that although the miR-122 binding site in HCV upregulates gene expression when residing in the 5’NCR of a replication-competent viral genome, it downregulates gene expression when located in the 3’NCR of a reporter mRNA, reminiscent of the outcome of a typical microRNA-mRNA interaction.
Fig. 2 Expression of reporter mRNAs containing miR-122 binding sites in their 3’ noncoding region. A: Diagram of capped Renilla luciferase-encoding reporter mRNA, expressed from plasmid pRL-SV40, and of firefly luciferase-encoding reporter mRNA, expressed (more ...)
Identification of a second, adjacent microRNA binding site in the HCV genome
To determine whether additional miR-122 binding sites increased the effect on reporter gene expression, a second copy of HCV sequence 1–45 was inserted in the 3’NCR of pLUC-122x1, resulting in the plasmid pLUC-122x2 (). As shown in the black bars in , sequestration of miR-122 by miR-122 antisense oligonucleotides resulted in an 80% increase in firefly luciferase activity compared to Random-2’OMe oligonucleotides. In contrast, ectopic expression of synthetic, wildtype miR-122 molecules caused a 70% decrease in luciferase production compared to mutant miR-122 p3-4 oligonucleotides (). These findings indicate that miR-122-mediated repression of target mRNAs can occur by binding of miR-122 to tandem target sites, and that the repression is greater than that mediated by a single target site.
Fig. 3 Expression of reporter mRNAs that contain tandem HCV sequence elements. A: Diagram of a capped firefly luciferase-encoding reporter mRNA, expressed from plasmid pLUC-122x2, which harbors two HCV sequence elements in its 3’ noncoding region. Shown (more ...)
To examine whether luciferase production in the chimeric mRNAs was modulated by direct binding of miR-122 to the tandem miR-122 binding sites, as opposed to binding to target sequences located in other mRNAs, the seed match sequences in both of the sites in pLUC-122x2 were mutated. Specifically, the nucleotides at positions 3 and 4 of each seed match sequence were mutated to their complement, i.e. U26C to A26G to yield S1:p3-4, such that the mutant sites are not predicted to bind wildtype miR-122 molecules. Instead, they should bind to mutant miR-122p3-4 molecules, restoring base pairing to the mRNA-miRNA pair. As shown by the striped bars in , miR-122 sequestration by methylated antisense miR-122 molecules still displayed an effect on reporter gene expression, although the effect was reduced relative to the effect observed with wildtype sequences. Furthermore, ectopic expression of either wildtype or mutant miR-122p3-4 microRNAs decreased reporter gene expression (). These results suggest that additional binding sites for wildtype miR-122 may be present in the reporter mRNAs. Indeed, inspection of HCV nucleotide sequences 1–45 revealed a potential second seed match sequence that could form a Watson-Crick base pair interaction with miR-122. This putative seed match 2, spanning viral nucleotides 38–43, is highlighted in .
To test whether both seed match 1 and 2 functioned in the first 45 nucleotides of the HCV genome, positions 3 and 4 in both sequence elements were mutated to their complementary nucleotides, i.e. U26C to A26G and U41C to A41G, to yield plasmid S1+S2:P3-4 (). Results from expression studies with this plasmid are shown by the white bars in . Neither sequestration of wildtype miR-122 nor overexpression of wildtype miR-122 had a significant effect on the expression of this reporter mRNA. However, ectopic expression of mutant miR-122p3-4 molecules caused an 80% decrease in luciferase activity. These findings provide genetic evidence for the presence of two functional miR-122 binding sites that can downregulate luciferase production.
Translational repression mediated by tandem miR-122 binding sites located in 3’ noncoding regions
There is some controversy about the mechanism by which microRNA-mediated repression occurs in mammalian cells. Studies have provided examples for microRNA-mediated repression at the translation initiation step, at the translation elongation step and at the post-elongation step. Furthermore, there are examples of microRNA-mediated mRNA degradation, possibly occurring as a result of translational repression (reviewed in (Jackson and Standart, 2007
; Nilsen, 2007
To examine the intracellular abundances of the reporter mRNAs, total RNA was extracted from Huh7 cells that had been transfected with plasmid pLUC-122x2 in the presence of the various small RNA modulators, and reporter mRNA abundances were quantified by real-time quantitative PCR. Whereas the ratio of firefly:Renilla luciferase activity changed with sequestration or overexpression of miR-122, as described above (, black bars), no significant change in the ratio of firefly:Renilla mRNA was observed under these conditions (). These findings argue that miR-122 mediates translational repression via two seed matches in the HCV genome when located in the 3’NCR of a luciferase mRNA, and that this repression likely occurs at a translational step and does not involve mRNA degradation.
Regulation of a tandem miR-122 binding site in the HCV genome by cellular miR-122
We have previously shown that mutation of seed match 1 diminished HCV RNA accumulation, which can be restored by mutant miR-122 molecules that can bind to the mutated seed match sequence (Jopling et al., 2005
). To examine a functional role for the seed match 2 site in modulating HCV gene expression, defined mutations at the p3 and p4 positions of the seed match () were introduced into a type 1a HCV genome that contains adaptive mutations that allow it to replicate in Huh7 cells (Yi et al., 2006
). In particular, double mutations (p3-4) in HCV were generated, in which seed matches 1 (S1) and 2 (S2) were both mutated at the p3 and p4 positions (). RNA abundance of mutant RNAs was measured after electroporation into Huh7 cells. The Northern blot in (lane 1) shows that significant amounts of wildtype HCV RNA could be detected 5 days after electroporation, whereas neither mutant S2:p3-4 nor S1+S2:p3-4 RNA could be detected in transfected cells (lanes 2 and 4). However, both S2:p3-4 and S1+S2:p3-4 RNA could be detected after co-transfection of miR-122p3-4 RNAs, predicted to base pair with the mutant HCV genomes (, lanes 3 and 5). Interestingly, ectopic expression of miR-122p3-4 repeatedly rescued the abundance of HCV RNAs that contained mutations at both S1 and S2 to near wildtype levels (, lane 5), suggesting that ectopically expressed miR-122 RNAs display cooperative effects on HCV gene expression. Therefore, miR-122 interaction at seed match 1 alone is not sufficient to augment HCV RNA abundance. We conclude that miR-122 interactions at both seed matches are required for efficient HCV RNA accumulation.
Fig. 4 Effects of mutations in seed match sequences on abundance of replication-competent HCV RNA. A: Diagram of the introduced mutations in the two viral seed match sequences for miR-122. B,C,D: RNA abundances of wildtype or mutant HCV RNAs. Abundances were (more ...)
To determine the effects of single-nucleotide mutations in seed match 1 and seed match 2, mutations at the p3 positions of both seed match sequences were tested. Similar to the findings with the double mutants, a single mutation in S2 (, lane 2) or single mutations in both S1 and S2 (, lane 4) abolished RNA accumulation, which could be restored by co-transfection of miR-122p3 molecules (, lanes 3 and 5). These findings also argue that miR-122 binding to seed match 1 is not sufficient to allow viral RNA amplification.
To examine whether both seed match sequences are required to maintain HCV RNA abundance and are occupied by miR-122 in a single HCV RNA molecule, we introduced distinct mutations into S1 and S2 and monitored rescue of RNA abundance after transfection of distinct miR-122 molecules. Specifically, a p3 mutation in S1 and a p3-4 mutation in S2 were generated in the same viral RNA molecule (). Transfection of this mutant RNA into Huh7 cells did not lead to detectable HCV RNA accumulation five days after electroporation (, lane 2). Presumably, the endogenous wildtype miR-122 RNA had no effect on either mutant seed match sequence. Co-transfection of either miR-122p3 or miR-122p3-4 molecules was not sufficient to restore RNA abundance (, lanes 3 and 4). In contrast, simultaneous transfection of both miR-122 mutant RNAs restored HCV RNA abundance (, lane 5). This experiment suggests that both miR-122 binding sites need to interact with miR-122, either sequentially or concurrently, in a single HCV molecule to ensure viral RNA amplification.
Effect of a miR-122 seed match substitution on HCV gene expression
Next, we examined whether roles for miR-122 can be substituted by other microRNAs. Thus, we exchanged the miR-122 seed match 1 sequence element in the HCV genome with a seed match element that is predicted to base pair with microRNA miR-21 (). Transfection of the mutant HCV-m21 genome into Huh7 cells failed to accumulate viral RNA (). While miR-21 is expressed at a five-fold lower level than miR-122 in these cells (data not shown), HCV RNA abundance could not be rescued after ectopic expression of miR-21 duplexes (data not shown). These findings suggest that oligomeric miR-122 complexes have specialized functions in HCV replication or that the seed match substitution affected sequences whose integrity are essential for viral genome amplification.
Fig. 5 Effect of a seed match substitution on abundance of replication-competent HCV RNA. A: Diagram showing the locations of the introduced seed match swap. B: RNA abundances of wildtype or mutant HCV-m21 RNAs after electroporation into Huh7 cells. An autoradiograph (more ...)
Modulation of viral gene expression by sequences encompassing the tandem miR-122 binding sites, including a conserved fourteen nucleotide spacer sequence element
To distinguish roles for sequences surrounding the miR-122 binding sites in microRNA binding or viral RNA replication, the replication-competence of HCV genomes carrying defined mutations was examined. It is known that nucleotide length between two tandemly located microRNA-binding sites can dictate the efficiency by which the microRNA regulates target mRNA expression (Doench and Sharp, 2004
; Saetrom et al., 2007
). In particular, optimal downregulation was found to be mediated by tandem microRNA binding sites in which the distance from the start of one seed to the start of the second seed sequence is 13–35 nucleotides (Saetrom et al., 2007
). A fourteen nucleotide distance was observed between S1 and S2 in HCV RNA (). Together with seed match sequences 1 and 2, this spacer sequence element is highly conserved between HCV genotypes, except that adenosine at position 36 is missing in genotypes 5 and 6 (). To examine whether the conserved linker sequence is important for miR-122-mediated regulation of HCV RNA abundance, conserved nucleotides C30 and C31 were changed to adenosines (). As shown in , transfected mutated viral genomes (pCC-AA) accumulated approximately two-fold less than wildtype RNA genomes (, lanes 2 and 3). Ectopic expression of miR-122 wt duplexes did not rescue viral RNA abundance. More dramatically, mutant viral RNAs with nucleotides C30 and C31 deleted (pΔCC) failed to accumulate to detectable levels after transfection into Huh7 cells (, lanes 4 and 5) and abundance could not be rescued by ectopic expression of miR-122 duplexes. Similarly, insertions of two or four uridines at position 32 () into the viral genome failed to generate significant levels of replication-competent RNAs (, lanes 3 and 4). Insertion of the pCC-AA substitution, or the pΔCC deletion, into the 3’NCR of pLUC-122x1 () displayed translational repression in a microRNA-dependent manner that was similar to pLUC-122x1-derived mRNAs which contain wildtype microRNA binding sites and spacer sequences (Suppl. Fig. 1
). These findings suggest that the spacer nucleotides contribute to the formation of a replication-competent RNA structure. Alternatively, engagement of the viral miR-122 binding sites with miR-122 has different effects on viral replication and reporter mRNA translation.
Fig. 6 Effects of mutations in spacer sequences on abundance of replication-competent HCV RNA. A: Diagram showing the locations of the introduced mutations in the spacer sequences. B,C: RNA abundances of wildtype or mutant HCV RNAs carrying mutations in the (more ...)
Fig. 7 MiR-122 seed match sequences and predicted interactions with miR-122. (A) Locations of the miR-122 seed match sequences (highlighted in red) in HCV genotype 1a. The nucleotides linking the two seed match sequences are highlighted in blue. The borders (more ...)