Structural requirements for the UBA domain of the mRNA export receptor Mex67 to bind its specific targets, the transcription elongation THO complex component Hpr1 and nucleoporin FXFG repeats. Maria Hobeika, Christoph Brockmann, Florian Gruessing, David Neuhaus, Gilles Divita, Murray Stewart, Catherine Dargemont To cite this version: Maria Hobeika, Christoph Brockmann, Florian Gruessing, David Neuhaus, Gilles Divita, et al.. Struc- tural requirements for the UBA domain of the mRNA export receptor Mex67 to bind its specific targets, the transcription elongation THO complex component Hpr1 and nucleoporin FXFG repeats.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2009, pp.17575-17583. 10.1074/jbc.M109.004374. hal-00394024 HAL Id: hal-00394024 https://hal.archives-ouvertes.fr/hal-00394024 Submitted on 28 May 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 26, pp. 17575–17583, June 26, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Structural Requirements for the Ubiquitin-associated Domain of the mRNA Export Factor Mex67 to Bind Its Specific Targets, the Transcription Elongation THO Complex Component Hpr1 and Nucleoporin FXFG Repeats*□S Received for publication, February 19, 2009, and in revised form, April 8, 2009 Published, JBC Papers in Press, April 28, 2009, DOI 10.1074/jbc.M109.004374 Maria Hobeika‡1,2, Christoph Brockmann§1,3, Florian Gruessing§, David Neuhaus§, Gilles Divita¶, Murray Stewart§, and Catherine Dargemont‡4 From the ‡Institut Jacques Monod, Universite´Paris VII, CNRS, 2 Place Jussieu, Tour 43, 75251 Paris Cedex 05, France, the §Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom, and the ¶CRBM-CNRS-UMR-2593, Universite´Montpellier I & II, Molecular Biophysics & Therapeutics, 1919 Route de Mende, 34293 Montpellier Cedex 5, France The ubiquitin-associated (UBA) domain of the principal Sac- (mRNPs)5 are intimately coupled (1, 2). This coordination of charomyces cerevisiae mRNA nuclear export factor, Mex67, can mRNA biogenesis is mediated by a diverse range of RNA-bind- bind both nuclear pore protein (nucleoporin) FG repeats and ing proteins and maturation enzymes (reviewed in Refs. 2 and Hpr1, a component of the TREX⅐THO complex that functions to 3). Moreover, during processing, the transcripts are under the link transcription and export. Using fluorescence resonance constant surveillance of RNA quality control mechanisms that energy transfer-based assays, we show here that Hpr1 and the ensure that only correctly processed mRNAs are exported to FG repeats interact with overlapping binding sites on the Mex67 the cytoplasm for translation (4–7). From yeast to human, UBA domain. We present the solution structure of the Mex67 translocation of fully mature mRNPs to the cytoplasm through UBA domain (UBA-Mex67) complexed with a FXFG nucleo- nuclear pore complex is thought to be mediated primarily by porin peptide and define residues engaged in the interaction and Mex67/NXF1 (also known as TAP), the primary mRNA export those involved in the FXFG-induced conformational change. receptor, which forms a heterodimer with Mtr2/NXT1 (P15) We show by NMR titration that the binding of Hpr1 produces that interacts directly with nuclear pore complex proteins (FG analogous changes in chemical shifts in similar regions of the nucleoporins) that have characteristic FG sequence repeats UBA domain. Together the data presented here indicate that (8–10). Previous work has shown that, like other nuclear trans- both Hpr1 and FXFG nucleoporins may bind in a similar way to port factors (reviewed in Refs. 11 and 12), the Mex67/TAP UBA the UBA-Mex67 domain. However, whereas binding of Hpr1 domain binds specifically to the two Phe rings of the FXFG allows UBA-Mex67 to interact with tetra-ubiquitin, the com- nucleoporin-binding motif (13, 14). Because Mex67/NXF1 has plex between UBA-Mex67 and FXFG is unable to bind mono- or low intrinsic affinity for mRNAs, it is recruited to the mRNP by tetra-ubiquitin, suggesting that both substrate binding and also several RNA-binding adaptors, including members of the the nature of the substrate may influence the affinity of the THO⅐TREX complex that couples transcription elongation to UBA-Mex67 domain for ubiquitin. mRNA export (2, 15, 16) as well as the TREX-2 complex, Yra1/ ALY and Sub2/UAP56 (reviewed in Ref. 3). Recent studies have shown that the ubiquitin pathway par- Transcripts generated by RNA polymerase II undergo a care- ticipates in the regulation of several key cellular functions fully orchestrated series of processing steps, including 5Ј cap- including mRNA nuclear export (17–19). Ubiquitin is a small ping, splicing, 3Ј end cleavage, and polyadenylation before 76-residue protein that can be covalently linked as a monomer being exported to the cytoplasm. The steps of mRNA biogene- or as a polyubiquitin chain to a lysine residue of a specific sub- sis leading to export-competent ribonucleoprotein particles strate (20, 21). This process, known as ubiquitylation, targets the substrate for a range of possible fates. The diverse functions of ubiquitylation are mediated through effector proteins that * This work was supported in part by a Program Grant from the Wellcome contain one or several ubiquitin-binding domains. Those Trust (to M. S.), by Agence Nationale pour la Recherche Grant BLAN06- domains are classified into different groups according to their 1_134099 (to C. D. and G. D.), and the Ligue contre le Cancer. □ structural fold, the best known of which is the ubiquitin-asso- S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2. ciated (UBA) domain (22, 23). Interestingly, the essential The atomic coordinates and structure factors (code 2KHH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformat- ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). 5 The abbreviations used are: mRNP, ribonucleoprotein particle; UBA, ubiq- 1 Both authors contributed equally to this work. uitin-associated; FRET, fluorescence resonance energy transfer; GST, gluta- 2 Supported by the Association de Recherche contre le Cancer. thione S-transferase; PBS, phosphate-buffered saline; dansyl, 5-dimethyl- 3 Supported by the Federation of European Biochemical Societies. aminonaphthalene-1-sulfonyl; FITC, fluorescein isothiocyanate; NOESY, 4 Equipe Labelise´e of the Ligue contre le Cancer. To whom correspond- nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single ence should be addressed: Institut Jacques Monod, 2 Place Jussieu, quantum coherence; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxym- Tour 43, 75251 Paris Cedex 05, France. Tel./Fax: 33-1-44276956; E-mail: ethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; TOCSY, [email protected]. total correlation spectroscopy. JUNE 26, 2009•VOLUME 284•NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 17575 This is an Open Access article under the CC BY license. Mex67 UBA Domain and Specific Target Binding mRNA export receptor Mex67/ NXF1 has a UBA domain at its C terminus that has been shown to interact with nucleoporin FG repeats (13). We recently reported that the UBA domain of Mex67 (UBA-Mex67) is not only required for mRNA nuclear export but also contributes to early co-transcrip- tional recruitment of the receptor to the mRNP (3, 24). In addition to showing that the Mex67 UBA domain was able to bind both ubiq- uitin and FG nucleoporins, our studies identified Hpr1 as a specific partner of the UBA domain of Mex67. Hpr1 is a component of the THO⅐TREX complex that links transcription on mRNA export and that is regulated by ubiquitylation in a transcription-dependent manner (25). We proposed that the interac- tion with the Mex67 UBA domain transiently protects Hpr1 from ubiquitin/proteasome-mediated de- gradation and thereby coordinates recruitment of the mRNA export machinery with transcription and early mRNP assembly (24, 25). With the exception of the UBA2 domain of HHR23A, most of the UBA domains studied to date have shown little or no substrate specific- ity (26–28). Therefore, because of its specific interactions with ubiq- uitinated Hpr1, the Mex67 UBA domain appears to be a promising model for understanding molecular basis of specific substrate recogni- tion by UBA domains. UBA domains share high structural homology and are characterized by the presence of three ␣ helices that are connected by two short loops, forming a compact fold with a hydrophobic core and surface hydrophobic patches implicated in protein-protein interactions. Struc- tural analysis of several UBA domains in complex with ubiquitin has indicated that UBA domains interact with ubiquitin mainly via the C terminus of helix 1, loop 1, and FIGURE 1. Hpr1 and FXFG repeats have comparable affinity for the UBA domain of Mex67. A, binding of helix 3 (29–31). The structure in FXFG to UBA-Mex67 (400 nM) was followed by measuring tryptophan emission at 340 nm upon excitation at solution of ubiquitin polymers var- 290 nm. B, binding of FXFG, FF18-Nsp1, and Hpr1-C to UBA-Mex67 was monitored by adding increasing con- ies according to the lysine residue centrations of FITC-labeled proteins (UBA-Mex67 partners) to dansyl-labeled UBA-Mex67 (400 nM) and meas- uring the quenching of dansyl fluorescence at 440 nm, upon excitation at 350 nm.