MicroRNA-mediated regulation of viral replication

Genes are long sections of DNA that are copied into long molecules of a similar substance, RNA. These messenger RNAs are interpreted by machines known as ribosomes to make proteins, which carry out the functions in our cells. Recently, it was found that some genes are copied to make very short sequences of RNA, known as microRNAs. MicroRNAs are not used to make protein themselves, but instead bind to messenger RNA molecules that have a matching sequence for the microRNA. This binding leads to a reduction in the amount of protein made from that messenger RNA, and so is important in controlling the levels of particular proteins present in a cell, and therefore the behaviour of the cell. The hepatitis C virus (HCV) genome is composed of a long strand of RNA, which enters liver cells where it is used as a template to make HCV proteins. These make more copies of the HCV RNA, a process known as viral replication. I recently found that miR-122, a microRNA that is only found in the liver, binds to a site in HCV RNA. This binding is needed for viral replication to occur. This positive effect on viral replication is very different to the negative effects on protein synthesis that miRNAs normally promote. Interestingly, if the miR-122 binding site from HCV is moved to a different place in a different gene, it acts to repress protein synthesis. The aim of this research is to understand how a microRNA can mediate two such different processes. As the location of the binding site is important for its function, versions of HCV will be made with sites in different locations and tested to see what the requirements for the site are. Proteins are known to be important for microRNAs to function, so proteins that bind to miR-122 while it interacts with HCV RNA will be detected. These will be compared to the proteins used by microRNAs to repress protein synthesis. Finally, experiments will be carried out to detect when in the HCV replication cycle miR-122 interacts. Together, these experiments will help to explain how miR-122 is able to regulate HCV replication. This research will be carried out in the RNA biology group in the new Centre for Biomolecular Sciences at the University of Nottingham. Researchers in the group work on several different aspects of RNA, and are based in state-of-the-art new laboratories with all the necessary facilities for RNA research.

The liver-specific microRNA-122 (miR-122) binds to a site close to the 5' end of hepatitis C virus (HCV) RNA and is essential for replication of the virus. miRNAs had previously been shown to act by binding to the 3' untranslated regions (UTRs) of target mRNAs and repressing gene expression. The miR-122 site from HCV acts to repress translation when inserted in the 3' UTR of a luciferase mRNA. Moreover, it was recently shown that several miRNAs bind to HCV RNA and repress gene expression. Therefore, it appears that the location of the miR-122 binding site in HCV is crucial in determining its function. In order to understand how a miRNA can mediate such different events, the mechanism by which miR-122 regulates HCV replication will be analysed and compared to miRNA-dependent repression of translation. 1. The requirements for location and context of the miR-122 binding site will be determined by mutational analysis, both in HCV and in the luciferase reporter construct in which miR-122 represses translation. This will allow comparison of the requirements for the two processes. 2. The protein factor requirements for the two processes mediated by miR-122 will be compared. A streptavidin affinity purification system will be used to detect proteins that interact with biotinylated miR-122 in complex with HCV RNA. Known and novel interacting proteins will be identified by western blotting and sequencing. RNAi will be used to determine the requirement for candidate proteins. 3. The relationship between miRNA binding and the HCV replication cycle will be studied. Direct binding of miR-122, and of miR-196, which represses HCV gene expression, to HCV RNA will be detected over the course of the replication cycle using strepatividin affinity purification followed by quantitative RT-PCR. Sucrose gradients will be used to determine whether miR-122 is associated with actively translating polyribosomes.