1. Introduction Hepatitis C Virus

2. Introduction microRNAs

3. Liver-specific MicroRNA-122 displays position dependent function

Christiane Brohm 27.10.2008

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-+ Ribavirin

E2E1

Lipid membraneNucleocapsid (core)

ss(+) RNA

Hepatitis C Virus (HCV) Profile

HCV Genome Organisation

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

Liver-specific microRNA: miR-122

Upregulation of HCV Replication

RISC complex

miR-122

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.

Identification of an additional binding site and it‘s effect on reporter-gene expression.

Position-Dependent Function for a Tandem MicroRNA

▪ 5‘NTR: upregulative function, increased viral RNA amount ▪ 3‘NTR: downregulative function, decreased viral RNA amount

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.

5‘ end insertion does not affect protein synthesis

Huh7 cells

pHCV.Luc+

Oligonucleotides

reporter gene assay

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

3‘ end insertion leads to decreased protein synthesis

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

Second, adjacent binding site for miR-122

Mutation of one binding site displays reduced effects

Mutation of both sites can only compensated by second site mutations in miR-122

→ Tandem sites mediate translational repression

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.

→ miR-122 interaction at both sites is required for efficient HCV RNA accumulation

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

- Genetic evidence for presence of 2 functional miR-122 binding sites

- Spacer sequences are highly conserved and contribute to the formation of a replication-competent RNA structure

- miR-122 function can not be substituted by other microRNAs

- Both miR-122 binding sites are required for HCV RNA accumulation in replication-competent genomes

- Regulation of protein synthesis is mediated by translational repression since mRNA amount were not reduced

- 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

Thank‘s for your attention!