The regulation of mRNA export by microRNAs

Ut Southwestern Medical Center, Dallas TX

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Abstract

DESCRIPTION (provided by applicant): The expression of eukaryotic genes depends upon proper processing and export of mRNAs from the nucleus. These events are facilitated by the association of nuclear proteins such as mRNA export factors with mRNA, which ensure that processed transcripts interact with and translocate through the nuclear pore complex (NPC). Because of its critical role in gene expression, mRNA export is a common target of viruses, such as vesicular stomatitis virus (VSV), that exploit the pathway to promote viral replication and down-regulate the host immune response. Recently, the regulation of gene expression by microRNAs (miRNAs) has been shown to play a role in the progression of VSV infection. In addition to viral infection, miRNAs regulate a variety of important pathways in the cell and are likely involved in the highly regulated process of mRNA export. However, the role of miRNAs in the regulation of post-transcriptional events such as mRNA processing and export remains largely uncharacterized. Thus, the overall goal of this proposal is to identify and characterize miRNAs that regulate mRNA export pathways and VSV-mediated mRNA export block. To identify miRNAs that are key regulators of mRNA export and VSV infection, a library of human miRNA mimics and inhibitors will be screened in human epithelial cells. Luciferase-based screening will be used to assess effects of miRNAs on bulk gene expression in the presence or absence of VSV infection. In addition, ATP levels will be measured as a surrogate for cell death to identify miRNAs that are essential for cell viability and that induce resistance or susceptibility to VSV-mediated cytoxicity. The miRNAs that inhibit bulk gene expression or confer susceptibility or resistance to VSV-mediated cells death will be subjected to a secondary screen using oligo-dT in situ hybridization to assess their effects on mRNA export. Upon identification of miRNAs, the targets of the most potent miRNAs that regulate mRNA export and VSV infection will be investigated using bioinformatics and proteomics approaches. The most potent regulators of mRNA export will be selected for characterization of the molecular mechanisms involved in miRNA regulation of mRNA export. Interacting partners of the selected targets will be identified biochemically and functional mRNA export assays will then be performed to address their role(s) within the mRNA export pathway. Together, these studies will identify and characterize miRNAs that regulate mRNA export and VSV infection, which may reveal additional layers of mRNA export regulation and novel virus-host interactions that may identify new aspects of viral pathogenesis. PUBLIC HEALTH RELEVANCE: The pathways involved in nuclear export of mRNAs are often disrupted in cancers and are also targeted by viruses, such as vesicular stomatitis virus (VSV). Importantly, VSV is an oncolytic virus that preferentially replicates in and kills cancer cells;therefore, it is currently being considered for cancer therapeutics. This project will focus on the identification and characterization of miRNAs that regulate mRNA export and VSV pathogenesis, which may allow the development of VSV strains with enhanced specificity for malignant cells and decreased general pathogenicity.