ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex

Abstract

The conserved TREX mRNA export complex is known to contain UAP56, Aly, Tex1, and the THO complex. Here, we carried out proteomic analysis of immunopurified human TREX complex and identified the protein CIP29 as the only new component with a clear yeast relative (known as Tho1). Tho1 is known to function in mRNA export, and we provide evidence that CIP29 likewise functions in this process. Like the known TREX components, a portion of CIP29 localizes in nuclear speckle domains, and its efficient recruitment to mRNA is both splicingand cap-dependent. We show that UAP56 mediates an ATP-dependent interaction between the THO complex and both CIP29 and Aly, indicating that TREX assembly is ATP-dependent. Using recombinant proteins expressed in Escherichia coli, we show that UAP56, Aly, and CIP29 form an ATP-dependent trimeric complex, and UAP56 bridges the interaction between CIP29 and Aly. We conclude that the interaction of two conserved export proteins, CIP29 and Aly, with UAP56 is strictly regulated by ATP during assembly of the TREX complex. . In yeast, the THO complex consists of four tightly associated subunits (Tho2, Hpr1, Mft1, and Thp1) (Piruat and Aguilera 1998;Jimeno et al. 2006). Likewise, the metazoan THO complex consists of a set of tightly associated proteins, three of which (fSAP79, fSAP35, and fSAP24; known now as THOC5, THOC6, and THOC7, respectively) do not appear to be conserved in yeast and two of which are orthologs of Tho2 (THOC2) and Hpr1 (THOC1) (Rehwinkel et al. 2004;Masuda et al. 2005). In yeast, Aguilera and coworkers (Piruat and Aguilera 1998) identified a protein known as Tho1 during the same genetic screen that they used to identify the THO complex. Subsequent characterization of Tho1 revealed that it functions in mRNP biogenesis and export, but this protein was not identified as a component of the THO/TREX complex (Piruat and Aguilera 1998;Jimeno et al. 2006). However, Tho1 is a multicopy suppressor of THO complex mutants and is recruited to mRNA in a THO complex-dependent manner (Piruat and Aguilera 1998;Jimeno et al. 2006). In humans, a counterpart of yeast Tho1 was identified based on sequence alignment (Jimeno et al. 2006). This protein, CIP29, was first reported as a cytokine-induced protein and later was linked to several cancers (Choong et al. 2001;Fukuda et al. 2002;Hashii et al. 2004;Leaw et al. 2004). Like yeast Tho1, CIP29 contains a SAF motif and binds to DNA, which led to the speculation that CIP29 functions in transcription (Aravind and Koonin 2000; Hashii et al. 2004).3 Corresponding author. E-MAIL rreed@hms.harvard.edu; FAX (617) 432-3091. Article is online at http://www.genesdev.org/cgi

Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

“…2B). It is also important to note that others have used translationally competent reporter pre-mRNAs as mRNA export reporters, providing further support for the green-shifted effect we observe (Galy et al 2004;Dufu et al 2010;Teplova et al 2011;Caporilli et al 2013).…”

Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability

“…Thus, inhibition of ATPase activity may generally yield dominant-negative effects across cellular functions. ATP is required for the interaction of UAP56/Sub2p with other factors involved in mRNA export (Dufu et al 2010;Folco et al 2012) and with RNA (Taniguchi and Ohno 2008). Since these activities predictably occur cotranscriptionally, failure to bind, or release, key interaction partners may be the trigger of transcription-dependent hyper-recombination, either via an inability to resolve DNA:: RNA hybrids or by increasing R-loop formation (Huertas and Aguilera 2003).…”

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

“…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] . ARS2 KD results in accumulation of mRNA in nuclear speckle domains [121] .…”

“…CBCA also plays an important role in mRNA export, largely mediated by the multisubunit transcription export (TREX) complex, which acts as an adaptor for the export receptor TAP/NXF1 [106][107][108][109][110][111] . Human TREX is composed of the THO complex (THOC1, THOC2, THOC5, THOC6, THOC7, Tex1), CIP29, UAP56 and ALY/REF [112][113][114] . TREX is involved in release of mRNA from nuclear speckle domains and coupling to the export receptor [115] .…”