Molecular Basis for the TREX-2 mRNA Nuclear Export Pathway - Project Summary Nuclear export of mRNAs through the nuclear pore complex (NPC) is an obligatory pathway for eukaryotic gene expression, which is regulated by various cellular stress conditions such as viral infections and immune responses. During gene expression, the TREX (TRanscription and EXport) complex is recruited co- transcriptionally and pre-mRNA processing further mediates TREX assembly on the transcript. Through the combined action of the TREX complex and other mRNA export factors, the major mRNA export receptor NXF1- NXT1 heterodimer is recruited to the mRNA. At the nuclear face of the NPC, NXF1-NXT1 bound to mRNA can either dock at the NPC or associate with an NPC-anchored complex termed TREX-2 (TRanscription and EXport 2) complex, enabling docking and translocation of mRNAs through the NPC. We and others have shown that the TREX-2 complex mediates nuclear export of a large subset of cellular mRNAs, among which are mRNAs that encode histone modifying enzymes and RNA processing factors, and influenza virus mRNAs. Additionally, we identified an RNA signal that can determine TREX-2 dependency. However, the molecular mechanisms involved in the functions of TREX-2 and the role of new constituents of the complex in the mRNA nuclear export pathway remain to be elucidated. Here, we will use interdisciplinary approaches including cell biology, biochemistry, and structural biology to pursue two extensive aims. In Aim 1, we will elucidate the differential role and interaction network employed by TREX-2 and a new form of the complex, TREX-2LENG8, to mediate nuclear export of cellular and influenza virus mRNAs. Additionally, we will investigate the RNA-protein interactions that determine TREX-2 or TREX-2LENG8 dependency. Thus, the findings from these studies will establish the molecular mechanisms by which TREX-2 or TREX-2LENG8 mediates recruitment and nuclear export of cellular and influenza virus mRNAs. In Aim 2, we will determine the structural and functional basis for the cellular NS1- BP protein in the TREX-2 mRNA export pathway. We have identified a network of interactions and potential mechanisms involving NS1-BP, NXF1-NXT1, and TREX-2 that mediate nuclear export of an important subset of cellular and influenza virus mRNAs. The latter also requires the viral NS1 protein. We will determine the structural basis for the established interactions and investigate their molecular mechanisms and functional significance within the TREX-2 mRNA export pathway. Moreover, we will systematically identify and characterize the cellular mRNAs that require NS1-BP for nuclear export and investigate the functional significance of NS1-BP interactions with NXF1-NXT1 and TREX-2 for nuclear export of these NS1-BP dependent mRNAs. Collectively, our studies will reveal fundamental knowledge on the role of the TREX-2 complex in mediating nuclear export of mRNAs, which impact a large subset of cellular mRNAs and influenza virus mRNAs. Additionally, the findings on the interaction between the influenza virus NS1 protein and the TREX-2 complex could be potentially used for designing antiviral strategies to inhibit influenza mRNA nuclear export and, consequently, viral replication.
