Gene expression requires processing of pre-mRNAs prior to the export of mature mRNAs from the nucleus. As a result, cells have evolved mechanisms to ensure that all pre-mRNA processing steps are completed prior to nuclear export. Such mechanisms involve the coupling of mRNA export with upstream steps in mRNA biogenesis, but little is known regarding factors or mechanisms that couple exports with pre-mRNA processing in mammalian cells. We and others have shown that the pathogenic NS1 protein of influenza A virus inhibits mRNA nuclear export, leading to down-regulation of host gene expression and inhibition of host antiviral response. NS1 additionally targets constituents of the polyadenylation machinery. Our preliminary data indicate that NS1 does not affect mRNA export and 3 processing independently, but instead targets a connection between these steps. Our central goal is to define this novel mechanistic connection between the polyadenylation and mRNA export machineries and uncover how it is targeted by influenza NS1. The application is a multi-PI proposal by Drs. Beatriz Fontoura and Nicholas Conrad, who have extensive expertise in mRNA export and 3-end formation, respectively. In addition, both PIs have considerable experience with host-viral interactions.
In Aim 1, we will determine the role of polyadenylation factors on the regulation of mRNA nuclear export.
In Aim 2, we will examine the effects of mRNA export factors on mRNA hyperadenylation and decay.
In Aim 3, we will determine the mechanisms of NS1-mediated inhibition of host gene expression at the interface of nuclear export and 3-end processing. We will use a combination of genetic, biochemical, and cell biological approaches including high-resolution imaging. In sum, we will investigate a novel mechanistic connection between polyadenylation and mRNA nuclear export and demonstrate how it is regulated by the host and by the influenza virus pathogenic factor NS1. Since this work focuses on cellular and viral mechanisms that regulate coupled export and processing and that disregulation of this interface is linked to pathogenesis, these findings may uncover new therapeutic strategies or targets.
The cellular processes studied here, messenger RNA processing and export, are relevant to many diseases, ranging from viral infections to cancer. In fact, one of the aims of this application is designed to uncover the function of a major virulence factor from influenza virus on a new interface between these cellular machineries that prevents host antiviral response.
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