The Proteins PDIP3 and ZC11A Associate with the Human TREX Complex in an ATP-Dependent Manner and Function in mRNA Export

The conserved TREX complex, which contains UAP56, Aly, CIP29, and the multi-subunit THO complex, functions in mRNA export. Recently, several putative new components of the human TREX complex were identified by mass spectrometry. Here, we investigated the function of two of these, PDIP3 and ZC11A. Our data indicate that both of these proteins are components of a common TREX complex and function in mRNA export. Recently, we found that both CIP29 and Aly associate with the DEAD box helicase UAP56 and with the TREX complex in an ATP-dependent manner. We now show that this is also the case for PDIP3 and ZC11A. Thus, together with previous work, our data indicate that the TREX complex participates in multiple ATP-dependent interactions.     

Chtop is a component of the dynamic TREX mRNA export complex

The TREX complex couples nuclear pre‐mRNA processing with mRNA export and contains multiple protein components, including Uap56, Alyref, Cip29 and the multi‐subunit THO complex. Here, we have identified Chtop as a novel TREX component. We show that both Chtop and Alyref activate the ATPase and RNA helicase activities of Uap56 and that Uap56 functions to recruit both Alyref and Chtop onto mRNA. As observed with the THO complex subunit Thoc5, Chtop binds to the NTF2‐like domain of Nxf1, and this interaction requires arginine methylation of Chtop. Using RNAi, we show that co‐knockdown of Alyref and Chtop results in a potent mRNA export block. Chtop binds to Uap56 in a mutually exclusive manner with Alyref, and Chtop binds to Nxf1 in a mutually exclusive manner with Thoc5. However, Chtop, Thoc5 and Nxf1 exist in a single complex in vivo. Together, our data indicate that TREX and Nxf1 undergo dynamic remodelling, driven by the ATPase cycle of Uap56 and post‐translational modifications of Chtop.     

Adaptor Aly and co‐adaptor Thoc5 function in the Tap‐p15‐mediated nuclear export of HSP70 mRNA

In metazoans, nuclear export of bulk mRNA is mediated by Tap‐p15, a conserved heterodimeric export receptor that cooperates with adaptor RNA‐binding proteins. In this article, we show that Thoc5, a subunit of the mammalian TREX complex, binds to a distinct surface on the middle (Ntf2‐like) domain of Tap. Notably, adaptor protein Aly and Thoc5 can simultaneously bind to non‐overlapping binding sites on Tap‐p15. In vivo, Thoc5 was not required for bulk mRNA export. However, nuclear export of HSP70 mRNA depends on both Thoc5 and Aly. Consistent with a function as a specific export adaptor, Thoc5 exhibits in vitro RNA‐binding activity and is associated with HSP70 mRNPs in vivo as a component of the stable THO complex. Thus, through the combinatorial use of an adaptor (e.g., Aly) and co‐adapter (e.g., Thoc5), Tap‐p15 could function as an export receptor for different classes of mRNAs.     

The THO/TREX complex functions in disease resistance in Arabidopsis

Powdery mildew pathogens are biotrophic fungi that infect large number of plant species. EDR1 (ENHANCED DISEASE RESISTANCE 1) is a negative regulator of plant disease resistance, and loss-of-function in the EDR1 gene confers enhanced disease resistance to powdery mildew pathogen Golovinomyces cichoracearum. In an edr1 suppressor screen, we recently found that a mutation in HPR1, a component of the THO/TREX complex, suppresses edr1-mediated disease resistance, however the hpr1 mutation enhances the ethylene-induced senescence in edr1. The hpr1 single mutant displays enhanced susceptibility, indicating that HPR1 is involved in plant defense responses.¹ THO/TREX is a conserved protein complex that functions in pre-mRNA processing and mRNA export. Several components of THO/TREX complex in Arabidopsis have been identified. By searching Arabidopsis database, we found that Arabidopsis (Columbia-0) has two copies of UAP56, another component of the THO/TREX complex, and the UAP56 proteins are highly conserved. Similar to human UAP56 protein, Arabidopsis UAP56 also localizes to the nucleus, showing a pattern similar to the splicing speckles. Further characterization of the components of THO/TREX in Arabidopsis will provide new insights into the role of THO/TREX in defense responses in plants.     

URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway

The TREX complex integrates information from nuclear mRNA processing events to ensure the timely export of mRNA to the cytoplasm. In humans, UAP56 and its paralog URH49 form distinct complexes, the TREX complex and the AREX complex, respectively, which cooperatively regulate the expression of a specific set of mRNA species on a genome wide scale. The difference in the complex formation between UAP56 and URH49 are thought to play a critical role in the regulation of target mRNAs. To date, the underlying mechanism remains poorly understood. Here we characterize the formation of the TREX complex and the AREX complex. In the ATP depleted condition, UAP56 formed an Apo-TREX complex containing the THO subcomplex but not ALYREF and CIP29. URH49 formed an Apo-AREX complex containing CIP29 but not ALYREF and the THO subcomplex. However, with the addition of ATP, both the Apo-TREX complex and the Apo-AREX complex were remodeled to highly similar ATP-TREX complex containing the THO subcomplex, ALYREF and CIP29. The knockdown of URH49 caused a reduction in its target mRNAs and a cytokinesis failure. Similarly, cytokinesis abnormality was observed in CIP29 knockdown cells, suggesting that CIP29 belongs to the URH49 regulated mRNA export pathway. Lastly, we confirmed that the export of mRNA in URH49-dependent pathway is achieved by NXF1, which is also observed in UAP56-dependent pathway. Our studies propose an mRNA export model that the mRNA selectivity depends on the Apo-form TREX/AREX complex, which is remodeled to the highly similar ATP-form complex upon ATP loading, and integrated to NXF1.     

Human mRNA export machinery recruited to the 5' end of mRNA

Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.     

Biochemical characterization of the ATPase and helicase activity of UAP56, an essential pre-mRNA splicing and mRNA export factor

DEXD/H-box protein UAP56 is an essential pre-mRNA splicing factor required for the first ATP-dependent spliceosome assembly step. UAP56 is also essential for the export of the majority of mRNAs from the nucleus to the cytoplasm. We performed biochemical characterization of UAP56's ATPase and helicase activity, which is important for further understanding the role of these activities in UAP56's function. We showed that UAP56 is an RNA-stimulated ATPase that can only hydrolyze ATP. We demonstrated that UAP56 is an ATP-dependent RNA helicase that can unwind substrates with 5' or 3' overhangs or blunt ends in vitro. We showed that U2AF(65) and Aly, two proteins known to interact with UAP56, do not influence UAP56's ATPase or helicase activity. We also demonstrated that several mutants in the conserved helicase motifs I, II, and III abolish UAP56's ATPase and/or helicase activity, providing tools for future investigation of the role of UAP56's ATPase and helicase activity in spliceosome assembly and mRNA export.