Identification of cellular targets of viral miRNAs

MiRNAs are important regulators of gene expression. Herpesviruses encode and express such miRNAs. Their molecular functions, however, remain elusive. When our project was started, monoclonal antibodies against the human Ago proteins were already available. We immunoprecipitated Ago2-associated miRNA targets from EBV- as well as KSHV-infected cells. We were able to validate a number of the identified targets as true miRNA targets demonstrating that our method is useful for the characterization of viral miRNA interaction networks in infected cells.
In addition, together with Project 4, we were able to identify target mRNAs in EBV-associated NK/T-cell lymphomas. We found that the pro-inflammatory cytokine IL1A is regulated by miR-142-3p and the oncogene BCL6 is regulated by miR-205.
mHV-68 serves as an important model organism for herpesvirus infection because it solely infects mouse cells. However, our antibodies were directed against the human proteins only. Therefore, we started to generate antibodies against the mouse Ago proteins (mAgo1-4). We have these antibodies in hands now and will extend our studies of viral miRNA interaction networks to the mHV-68 system. In addition, we have identified in close collaboration with subproject 3 novel mHV-68 miRNAs. Interestingly, these novel molecules are generated from unusual precursor molecules by a non-canonical mIRNA biogenesis pathway.
With the identification of EBV as well as KSHV miRNA targets and the generation of monoclonal antibodies against mouse Ago proteins we have clearly reached the main goals of our project. We have performed CLIP-assays (Ago-mRNA crosslinking by UV light) and identified EBV-miRNA targets. The results are currently analyzed in close collaboration with Project 1.
With the establishment of the monoclonal antibodies against all four human Ago proteins, we have completed one of our major goals. The antibodies are specific and allow for the identification of Ago-specific binding partners such as target mRNAs. We have extended our work and generated a peptide tool that allows for the isolation of all Ago proteins at once. It is also not restricted to an organism and can be broadly used. It is very likely that this peptide will become an important tool in the entire miRNA field.