Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Abstract

REF/Yra1p interacts with TAP/Mex67p, and in yeast this interaction leads to displacement of Sub2p from Yra1p (7). TAP heterodimerizes with p15 and binds nucleoporins through central and C-terminal domains (8), directing the mRNP to the nuclear pore and promoting transport to the cytoplasm. On the cytoplasmic side of the nuclear pore, Dbp5p triggers displacement of Mex67p from mRNA. Yra1p binds mRNA early during its nuclear maturation but is no longer bound once it reaches the nuclear periphery (9). Consistent with this finding, analysis of Balbiani ring pre-mRNPs shows that UAP56 and REF accompany the mRNP to the nuclear periphery where UAP56 and then REF dissociate during translocation through the pore (10).Although Yra1p is essential for yeast mRNA export, depletion of REF in higher eukaryotes does not block bulk mRNA export (11), suggesting that other proteins can fulfill this role and that there may be functional redundancy between export adaptors. The shuttling SR proteins 9G8, SRp20, and SF2/ASF directly bind TAP by short arginine-rich peptides (12, 13) and can function as export factors (14). Even in yeast, other proteins can recruit Mex67p to the mRNP, including Yra2p (15) and Npl3p.The fact that TAP binds RNA weakly in vitro led to the idea that export factors such as REF, which bind RNA avidly, bridge the interaction between TAP and mRNA, leading to the term mRNA export adaptors (15). However, both TAP and Mex67p are readily UV-cross-linked to mRNA in vivo (16)(17)(18), suggesting a direct stable interaction at some point during export. Here, we show that mRNA is handed over from export adaptors to TAP and that at least in vitro, export adaptors have the ability to enhance the RNA-binding activity of TAP. ResultsThe RNA-and TAP-Binding Sites on REF Overlap. In Saccharomyces cerevisiae, Mex67p binding to Yra1p triggers displacement of Sub2p (7), so we established whether this is the case for the mammalian orthologs. We examined whether UAP56 coimmunoprecipitated (Co-IP) with REF2-I (REF) in the presence of increasing amounts of TAP. This analysis revealed that TAP triggered dissociation of UAP56 from REF (Fig. 1A, lanes 5 and 6).We analyzed organization of the resulting REF-TAP-RNA ternary complex by examining how REF binds RNA. NMR

Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry

“…A FLAGtagged REF2-I construct was transiently transfected into 293T cells, purified under stringent conditions using FLAG-agarose as previously described [33]. In each case the protein purifications were analyzed using SDS PAGE confirming proteins were purified to greater than 90% (see Supplementary Figure 1, which can be found in the electronic version of this article).…”

SRSF1-dependent nuclear export of C9ORF72 repeat transcripts: targeting toxic gain-of-functions induced by protein sequestration as a selective therapeutic strategy for neuroprotection

“…Successive rounds of DDX39B-dependent ATP hydrolysis were suggested to facilitate the assembly of TREX by loading of proteins with nuclear export adaptor functions [21] . Mutually exclusive interactions of ALYREF with DDX39B and the nuclear export factor 1 (NXF1, also known as TAP [23] ) lead to the displacement of DDX39B and handover of the RNA molecule from the nuclear export adaptor to NXF1 by switching NXF1 to a high RNA affinity mode [24] following remodeling of NXF1 into an open conformation that exposes its RNA-binding domain [25] . The assembly of TREX and remodeling of NXF1 is much more complex than described here, involving additional proteins, such as nuclear export co-adaptors, post-translational modifications and dynamic re-arrangements (recently reviewed in [26,27] ).…”

“…On the cytoplasmic side of the NPC, activation of the DEAD-box RNA helicase Dbp5 activity (DDX19B in human) by interaction with the nucleoporin GLE1 (RNA export mediator) associated to inositol hexakisphosphate (IP6) leads to recruitment of Dbp5/DDX19B onto the mRNAs promoting the unwinding of secondary RNA structures, the displacement of bound protein factors and the cytoplasmic release of mRNAs (reviewed in [31] ). Dissociation of NXF1 from the RNA and the nuclear export adaptor is predicted to revert NXF1 into a low RNA affinity mode that would also contribute to efficient mRNA release [24,32] . It remains unknown whether the CAP-dependent translation of mRNAs is linked or not to the nuclear export process and/or to the cytoplasmic release.…”

The Diverse Requirements of ARS2 in nuclear cap-binding complex-dependent RNA Processing

“…The presence of the exon junction complex (EJC), which is deposited following splicing, stimulates TREX recruitment [106,107,110,112,116] , ensuring correctly processed transcripts are exported. Following binding of TREX subunits to mRNA, ALY/REF and THOC5 handover the mRNA to the TAP/NXF1-p15 heterodimer [117] , which then transports the mRNA through the nuclear pore [111,118] . ALY/REF directly interacts with CBP20/80 [104,119,120] , while THOC2, UAP56, and CIP29 independently interact with ARS2 [113] , and these interactions are required for efficient mRNA export [120,121] .…”