Gene expression networks typically evolve multiple layers of regulatory coordination to facilitate coherent response to developmental, physiological or environmental conditions. Transposition of mobile elements, including endogenous retroviruses, has been hypothesized to create or modify gene regulatory networks, particularly through effects on transcriptional initiation. Some elements are also known that affect alternative RNA processing events, potentially allowing post-transcriptional coordinating mechanisms that may be subject to natural selection. We previously showed that the major allele of nuclear export factor Nxf1 in Mus musculus castaneus mice is a semi-dominant suppressor of de novo mutations caused by insertion of intracisternal A particle (IAP) endogenous retroviruses into host gene introns, by mitigating IAP-induced alternative RNA processing. The molecular phenotype of Nxf1-mediated suppression includes both an increased level of correctly processed host gene transcript and a decrease in virus- induced alternative forms. This was unexpected, as biochemical data for Nxf1 suggest that its major activity is export of ribonucleoprotein particles after completion of RNA processing. However, many viruses also attack Nxf1 to promote viral gene expression over that of the host. The genetic modifier activity of Nxf1 resides in a single amino acid substitution, E610G, that appears to be under positive directional selection in wild mice and that coordinately affects several IAP-inserted host genes in laboratory mice. These results suggest unexpected mechanisms by which Nxf1 regulates gene expression beyond its known role in export. Several Nxf1 binding partners are known to influence transcriptional elongation rate in other contexts and elongation rate influences alternative RNA processing steps. To understand the mechanism and the unexpected plasticity in a pathway that is conserved among animals and fungi, we propose three aims. (1) We will test the interdependence of suppression, elongation rates, and epigenetic marks related to elongation and retroviral silencing. (2) We will determine the effect of suppressing Nxf1 variants on protein complex formation both in intact cells and in simplified in vitro assays and test the role of variant partners in regulated suppression. (3) We will quantify the effects of each Nxf1 residue on cell viability, genetic suppression, and interactions with key partners using a deep mutational scanning approach.
Public Health Relevance: Understanding basic mechanisms through which gene expression networks have been modified by selective pressures and can be manipulated to favor host gene expression programs over those of pathogens, including RNA viruses, and molecular parasites such as retrotransposons, has potential applications in infectious disease, stem cell biology, and cancer. Understanding the network architecture and properties of Nxf1, an unexpected genetic modifier of retrovirus-induced mutations, will provide new insight into basic mechanisms of RNA processing and have application in testing expression level effects in mouse models of genetic disease.
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