Introduction Hepatitis C Virus • Introduction microRNAs • Liver-specific MicroRNA-122 displays position dependent function Christiane Brohm 27.10.2008
Hepatitis C Virus (HCV) Profile E1 Family: Flaviviridae Genus: Hepacivirus Species: Hepatitis C virus (6 genotypes) Size: 50-60 nm Genome: (+) ssRNA, ~9.6 kb Prevalence: 130 million patients Therapy: PEG-IFN-a + Ribavirin E2 ss(+) RNA Nucleocapsid (core) Lipid membrane
MicroRNA Mechanism of Action ▪ 21-22nt RNA molecule ▪ Expression of microRNAs in wide range of eukaryotic organisms ▪ Inhibition of mRNA expression by translation repression or mRNA cleavage
RISC complex miR-122 Upregulation of HCV Replication Liver-specific microRNA: miR-122
Position-Dependent Function for a Tandem MicroRNA Aim of the study Analysis of miR-122 function by binding either the 5‘NTR or 3‘NTR. Importance of the location of the miR-122 binding site for mRNA regulation. ▪ 5‘NTR: upregulative function, increased viral RNA amount ▪ 3‘NTR: downregulative function, decreased viral RNA amount Identification of an additional binding site and it‘s effect on reporter-gene expression.
pHCV.Luc + Oligonucleotides reporter gene assay Huh7 cells 5‘ end insertion does not affect protein synthesis Insertion of complete HCV 5‘NTR in reporter mRNA. After sequestration of miR-122 no downregulation of protein synthesis Synthetic miR-122 did not affect reporter-gene expression in contrast to effects seen with replication-competent HCV genomes.
3‘ end insertion leads to decreased protein synthesis Insertion of miR-122 binding site at the 3‘ end. After sequestration of miR-122 upregulation of protein synthesis Synthetic miR-122 expression induced decreased reporter-gene expression
→ Tandem sites mediate translational repression Second, adjacent binding site for miR-122 Second copy of binding site was inserted in 3‘ end of reporter mRNA. Binding of miR-122 to tandem sites induces a stronger effect on reporter gene expression Mutation of one binding site displays reduced effects Mutation of both sites can only compensated by second site mutations in miR-122
→ miR-122 interaction at both sites is required for efficient HCV RNA accumulation Examination of functional role for the second binding site in HCV gene expression Combination of mutations in either one or both binding sites in a replication-competent HCV genome.
Can miR-122 function be substituted by other microRNAs? Exchange of miR-122 binding site with miR-21 binding site in 5‘NTR. → miR-122 complexes have specialized functions in HCV replication or → Substitution of binding site affects sequences essential for genome amplification
Role of spacer sequences surrounding binding sites Do they have an effect on miR-122 binding or viral genome amplification? → Spacer regions (14nt) are highly conserved among HCV genotypes → Contribution to the formation of a replication-competent RNA structure
Summary - Insertion of miR-122 binding site in 5‘ end of reporter genomes did not lead to altered protein synthesis. - Whereas 3‘ end insertion induces decreased reporter gene activity, reminiscent of the outcome of a typical microRNA-mRNA interaction - Genetic evidence for presence of 2 functional miR-122 binding sites - Regulation of protein synthesis is mediated by translational repression since mRNA amount were not reduced - Both miR-122 binding sites are required for HCV RNA accumulation in replication-competent genomes - miR-122 function can not be substituted by other microRNAs - Spacer sequences are highly conserved and contribute to the formation of a replication-competent RNA structure