Kurzfassung

MicroRNAs (miRNAs) are genome-encoded ~22 nucleotide non-coding RNAs that associate with the RNA-induced silencing complex (RISC) in order to mediate posttranscriptional silencing of gene expression. MiRNAs play critical roles during cellular differentiation and are associated with inflammatory diseases and cancer. Because viruses have evolved strategies to manipulate their host cells to promote viral replication, they have often served as tools to discover and characterize fundamental...MicroRNAs (miRNAs) are genome-encoded ~22 nucleotide non-coding RNAs that associate with the RNA-induced silencing complex (RISC) in order to mediate posttranscriptional silencing of gene expression. MiRNAs play critical roles during cellular differentiation and are associated with inflammatory diseases and cancer. Because viruses have evolved strategies to manipulate their host cells to promote viral replication, they have often served as tools to discover and characterize fundamental cellular pathways. We have recently discovered the first mammalian virus encoded inhibitor of cellular miRNA function. We could show that the poxviral poly(A) polymerase VP55 that polyadenylates viral transcripts also induces the global poly(A)-tailing of host miRNAs which results in their rapid degradation. This first demonstration of viral antagonism of host miRNAs has raised several important questions about the relevance of cellular miRNA pathways during infection and the evolution of viral strategies to counteract and to exploit these pathways. To adress these questions, we have established VP55-mediated degradation of miRNAs as a tool to study the role of host miRNAs during RNA virus infection. Here, we propose to molecularly dissect the various functions of miRNAs during infection and to exploit VP55 induced miRNA degradation in order to identify cellular miRNA degradation pathways critical for the regulation of gene expression.  In Aim 1 we will generate a system that allows poxvirus replication in the presence of functional cellular miRNAs. To this end, we will perform mutagenesis on VP55 in order to identify mutants that are specifically impaired in their ability to polyadenylate miRNAs while still maintaining full mRNA polyadenylation capacity. Additionally, we will express the methyltransferase Hen1 during viral infection. Hen1 specifically 2’-O-methylates miRNAs at the 3’end, a modification that renders miRNAs insensitive to VP55-mediated degradation. In Aim 2 we will define the interactome of VP55 by performing mass spectrometry and co-immunoprecipitation analysis. These studies aim at the identification of the cellular miRNA degradation pathway which is currently unknown and is anticipated to have broad relevance during cellular differentiation and proliferation and, therefore, also for inflammation and cancer. These studies will significantly increase our understanding of the functions of the host miRNA silencing and degradation machinery and thereby facilitate the identification of novel molecular targets for the therapy of infection, inflammation and cancer. » weiterlesen» einklappen