The ubiquitin pathway regulates multiple steps of Mex67-mediated mRNA export

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Thesis

Reference

The ubiquitin pathway regulates multiple steps of Mex67-mediated mRNA export

IGLESIAS, Nahid

Abstract

Dans la levure et chez les eucaryotes supérieurs, les ARN messagers (ARNm) matures sont exportés du noyau vers le cytoplasme par Mex67-Mtr2 (TAP-NXF1 chez les eucaryotes supérieurs), le principal récepteur d'export des ARNm. La faible affinité de ce dernier pour les ARNm implique le besoin d'un adaptateur, Y ra1/REF, seul adaptateur connu de Mex67/TAP jusqu'alors. Ce travail a identifié deux nouveaux adaptateurs pour le recrutement de Mex67 sur les ARNm et a demontré la relevance fonctionnelle de ces interactions pour l'export des ARNm. De plus, nous avons mis en évidence l'importance de la modification post-traductionnelle par l'ubiquitination dans l'export des ARNm. Enfin, nous avons découvert qu'hormis son rôle dans l'export des ARNm, Mex67 avait d'autres fonctions, comme coordonner le recrutement de la machinerie d'export à la transcription et aux étapes précoces de la biogenèse des ARNm, ainsi que d'ancrer les gènes au pore nucléaire.

IGLESIAS, Nahid. The ubiquitin pathway regulates multiple steps of Mex67-mediated mRNA export. Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3955

URN : urn:nbn:ch:unige-21644

DOI : 10.13097/archive-ouverte/unige:2164

Available at:

http://archive-ouverte.unige.ch/unige:2164

Disclaimer: layout of this document may differ from the published version.

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The Ubiquitin Pathway Regulates Multiple Steps of Mex67-mediated mRNA Export

THÈSE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Nahid IGLESIAS de

Pully (VD)

Thèse N° 3955

GENÈVE

Atelier de reproduction de la Section de physique

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The Ubiquitin Pathway Regulates Multiple Steps of Mex67-mediated mRNA Export

THÈSE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Nahid IGLESIAS

de Pully (VD)

Thèse N° 3955

GENÈVE

Atelier de reproduction de la Section de physique

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between January 2003 and February 2008 and was supported by the Département d’Instruction Publique (DIP) de Genève. Essential parts of this work are published in the following papers, listed in chronological order:

1. Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription. C Gwizdek*, N Iglesias*, MS Rodriguez, B Ossareh-Nazari, M Hobeika, G Divita, F Stutz, C Dargemont. Proc Natl Acad Sci U S A. 31;103(44):16376- 81. *equal contribution. (Oct 2006).

2. Co-transcriptional recruitment of the mRNA export receptor Mex67 contributes to nuclear pore anchoring of activated genes. G Dieppois*, N Iglesias*, F Stutz. Mol Cell Biol. 26(21):7858-70. *equal contribution. (Nov 2006)

3. A new role for the poly(A)⁺ binding protein Nab2 in Mex67-induced mRNA export.

N Iglesias, P Vinciguerra, AH Corbett and F Stutz. To be submitted.

In addition, other research projects that were not included in this thesis but in which I was involved during the course of my PhD are presented in Annex:

4. Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export. P Vinciguerra, N Iglesias, J Camblong, D Zenklusen, and F Stutz. EMBO J.

23;24(4):813-23. (Feb 2005)

5. Coordination of Hpr1 and ubiquitin binding by the UBA domain of the mRNA export factor Mex67. M Hobeika, C Brockmann, N Iglesias, C Gwizdek, D Neuhaus, F Stutz, M Stewart, G Divita, and C Dargemont. Mol Biol Cell. 18(7):2561-8. (May 2007)

6. Anti-sense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae. J Camblong, N Iglesias, C Fickentscher, G Dieppois, and F Stutz. Cell. 131(4):706-17 (Nov 2007)

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I want to start by thanking Françoise Stutz, who coordinated my graduate studies. The great interest with which she supervised me and the support she offered, with much patience. The trust she invested in me helped me gain confidence and motivated me to always explore new approaches and research directions.

In addition, I would like to thank Dr. Catherine Dargemont and Prof. Anita Corbett, as well as members of their labs, for fruitful collaboration and precious advices.

I also thank Prof. Ed Hurt and Prof. Didier Picard for accepting to review my thesis and to be members of the jury for my defense.

I am grateful to all past and present members of the Stutz lab for making the past years intellectually stimulating as well as fun. Nissrine for all that we have shared during these last months and Guennaëlle for her good spirit. A special thanks for Patrizia, who had not only taught me a variety of techniques, but also supported me during good and bad times of my PhD thesis with her professional and personal advice for which I am indebted.

I would like to thank all the people from the BICEL department for their help and kindness.

I would further like to thank all my friends for the time we share together and their support. A special big thank to my best friends Caroline, Magalie, Patrizia and Delphine for always taking me out on adventures, big as small, to be always willing to lend a ear to me, and for helping me realize that there are more things in life than science.

My parents, my brother and my aunt have laid the foundation for my career, and without their love and support, I could not have realized my purposes. Many thanks.

Finally, my last thank goes to Romain, for all his love, strength and enormous support. I couldn’t have done all this work without the energy he gave me!

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La compréhension des différents mécanismes moléculaires responsables de l'expression des gènes a été un intérêt central des biologistes moléculaires depuis plusieurs décennies. L’étape initiale de l’expression des gènes, la transcription, consiste à convertir dans le noyau l’information génétique en ARN messagers (ARNm) qui seront exportés dans le cytoplasme pour être traduits en protéines. Étant donné que l’enveloppe nucléaire est une entrave physique entre la transcription et la traduction chez les cellules eucaryotes, l’export des ARNm est un processus central dans l’expression des gènes et, par conséquent, essentiel pour la viabilité des cellules.

Dans la levure et chez les eucaryotes supérieurs, les ARNm matures sont exportés vers le cytoplasme par le récepteur de transport nucléaire conservé Mex67-Mtr2 (TAP-p15 chez les métazoaires). Lorsque cette étude a été initiée, les modèles proposaient que le complexe THO (constitué d’Hpr1, Mft1, Thp2, et Tho2), associé à la machinerie de transcription, assurait le recrutement de Yra1/REF et de son partenaire Sub2/UAP56 sur les transcrits naissants. THO, Yra1/REF, et Sub2/UAP56 constituent le complexe TREX, couplant la transcription et l’export. Une fois la biogenèse et la maturation des ARNm accomplie, Mex67/TAP serait recruté sur les ARNm devenus matures par le biais de Yra1/REF, qui agirait comme une protéine adaptatrice. L’association de Mex67/TAP à Yra1/REF dissocierait Sub2/UAP56 des ARNm et permettrait par la suite leur export au travers des pores nucléaires grâce à l’interaction directe entre Mex67/TAP et les nucléoporines formant les pores.

Cependant, Yra1/REF n’est probablement pas le seul adaptateur pour le recrutement de Mex67/TAP sur les ARNm. En effet, (i) Yra1 ne lie pas tous les transcrits de la levure;

(ii) Des mutations dans la protéine Yra1 qui abolissent son interaction avec Mex67 in vitro, réduisent mais n’éliminent pas totalement l’association entre Mex67 et les ARNm;

(iii) Yra1/REF n’est pas essentiel pour l’export des ARNm ni chez la Drosophile ni chez C. elegans.

L’objectif principal de ce projet de thèse visait à déchiffrer les mécanismes à la base du recrutement de Mex67/TAP sur les ARNm en utilisant la levure Saccharomyces cerevisiae comme organisme modèle. Dans la première partie de ce projet (section 2.1),

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domaine C-terminal UBA (pour « associé à l’ubiquitine »), et que cette association précoce était importante pour le bon fonctionnement de Mex67 dans l’export des ARNm.

En collaboration avec le groupe du Dr. Catherine Dargemont (Paris), un crible double hybride a identifié Hpr1, une protéine récemment identifiée comme étant ubiquitinée co- transcriptionnellement, comme partenaire de Mex67. Finalement, nous avons montré qu’Hpr1 contribue au recrutement co-transcriptionnel de Mex67 et que sa liaison transitoire au domaine UBA de Mex67 le protège de la dégradation par le protéasome.

Ainsi, le domaine UBA de Mex67 coordonne le recrutement de la machinerie d’export à la transcription et aux étapes précoces de la biogenèse des ARNm.

La seconde partie (section 2.2) décrit des résultats, obtenus avec Guennaëlle Dieppois, doctorante dans le laboratoire du Dr. Françoise Stutz. Notre étude a montré que Mex67 et Mlp1, une protéine associée à la face nucléaire du pore, étaient impliqués dans l’ancrage des gènes à la périphérie. De plus, nous avons trouvé que l’activation de la transcription et non la production d’ARNm jouait un rôle dans l’ancrage des gènes étudiés aux pores.

Dans la dernière partie de ce travail (section 2.3), nous avons cherché à définir si Nab2, une protéine se liant aux queues poly(A)⁺ des ARNm, était un adaptateur alternatif pour le recrutement de Mex67 sur les ARNm. Nous avons démontré que Mex67 se liait directement à Nab2, et que Yra1 stimulait fortement cette interaction. De plus, la présence de Yra1 n’est plus obligatoire pour la viabilité des cellules lorsque Nab2 est exprimé en excès. Nous avons également découvert que Yra1 était ubiquitiné par l’E3 ligase Tom1, et que l'ubiquitine régulait la dynamique du complexe trimérique formé par Nab2, Mex67 et Yra1. En conclusion, nous avons constaté que Yra1 devait se dissocier des ARNm nucléaires pour permettre leur export vers le cytoplasme. De façon générale, ce travail prouve que Yra1 n'est pas un adaptateur pour Mex67, comme précédemment pensé, mais plutôt un co-facteur, contrôlé par l'ubiquitine, qui régulerait aussi bien l'association de Mex67 avec Nab2, son nouvel adaptateur, que l’export des ARNm.

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Understanding the different molecular mechanisms responsible for gene expression has been a central interest of molecular biologists for several decades. Transcription, the initial step of gene expression, consists in converting the genetic code into a dynamic messenger RNA (mRNA) that will specify a required cellular function following translation into protein. As the nuclear envelope poses as a physical barrier between transcription (in the nucleus) and translation (in the cytoplasm) in eukaryotic cells, the nuclear exit of mRNAs is a central process in gene expression and hence essential for cell viability.

From yeast to human, mRNA export is achieved through the binding on mature mRNAs of an evolutionarily conserved machinery, minimally defined by the mRNA export receptor heterodimer Mex67-Mtr2 (TAP-p15 in metazoans). When this work was initiated, the current view of this process was proposing that THO complex (consisting of Hpr1, Mft1, Thp2, and Tho2) associated with the transcription machinery loads Yra1/REF and its partner Sub2/UAP56, an ATPase RNA helicase, onto nascent transcripts. THO, Yra1/REF, and Sub2/UAP56 constitute the TREX complex, proposed to couple transcription and export. After completion of mRNA biogenesis and processing, Yra1/REF acts as an adaptor protein for Mex67/TAP recruitment on mature mRNAs. This event displaces Sub2/UAP56 from Yra1/REF. Mex67/TAP directly binds nucleoporins, thus targeting the mature mRNAs to and through the nuclear pore, resulting in protein synthesis in the cytoplasm.

However, Yra1/REF is most likely not the only adaptor protein for Mex67/TAP recruitment on yeast and metazoan mRNAs. Evidence for such speculation is that (i) Yra1 does not bind to all yeast transcripts; (ii) Yra1 mutants defective in Mex67 binding in vitro reduce, but do not eliminate, association of Mex67 with mRNAs; and (iii) Yra1/REF is not essential for mRNA export neither in Drosophila nor in C. elegans.

The main objective of this PhD project aimed to decipher the mechanisms underlying Mex67/TAP recruitment on mRNAs using the yeast Saccharomyces cerevisiae (S.

cerevisiae) as a model system. In the first part of this project (section 2.1), we sought to define when and how Mex67 associates with the newly formed mRNAs. Notably, we

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recruitment and that this early event is important for proper mRNA export function. As part of a collaboration with the group of Dr. Catherine Dargemont (Paris), a two-hybrid screen identified Hpr1, recently described to be ubiquitinated co-transcriptionally, as an interacting partner of the Mex67-UBA domain. Finally, we obtained evidence that the ubiquitination of Hpr1 contributes to the co-transcriptional recruitment of Mex67 and that the binding of Mex67-UBA transiently protects Hpr1 form degradation by the proteasome. Mex67 dissociation may promote Hpr1 degradation and THO complex recycling. In summary, our analysis proposes that the Mex67-UBA domain coordinates the recruitment of the mRNA export machinery with transcription and early steps in mRNA biogenesis.

The second part of this thesis (section 2.2) describes results that were obtained together with Guennaëlle Dieppois, PhD student in our lab. We aimed to find mechanistic insights of nuclear pore complex (NPC)-gene anchoring using the combination of LacO/LacI system and chromatin immunoprecipitation (ChIP) experiments. This work identified Mex67 and the NPC-associated protein Mlp1 as proteins required for gene to pore anchoring and provide evidence that transcription activation but not mRNA production plays a major role in NPC anchoring of the studied genes.

In the last part of this work (section 2.3), we investigated whether the poly(A)⁺ binding protein Nab2 is an alternative adaptor for Mex67 recruitment on mRNAs. We demonstrated that Mex67 directly binds to Nab2 and bypasses Yra1 function when over- expressed. A competition experiment further showed that Mex67, Yra1 and Nab2 form a trimeric complex. Moreover, we uncovered that Yra1 is ubiquitinated by the ubiquitin E3 ligase Tom1, and that ubiquitin regulates the dynamics of the trimeric complex. Finally, we found that Yra1 has to dissociate from nuclear mRNAs to allow their exit to the cytoplasm. Overall, this study shows that Yra1 is not an adaptor for Mex67 as previously thought, but rather a co-factor, controlled by ubiquitin, that regulates Mex67 association with its new adaptor Nab2 as well as mRNA export.

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3.3 Post-translational modifications as a mean of tight regulation in mRNA export 124 3.4 Role of Mex67 early recruitment in mRNA biogenesis and export 126 3.5 Role of Mex67 and nascent RNA in gene anchoring to nuclear periphery 128 3.6 Is Mex67 recruitment on transcribing genes the cause or the consequence of gene

anchoring? 130

3.7 Functional significance of gene anchoring 131

3.8 Concluding Remarks 135

4 ANNEXES 136

4.1 Perinuclear Mlp proteins downregulate gene expression in response

to a defect in mRNA export. EMBO J., 2005, 23;24(4):813-23. 137

4.2 Coordination of Hpr1 and ubiquitin binding by the UBA domain of the mRNA export factor Mex67. Mol Biol Cell. 2007, 18(7):2561-8. 165

4.3 Anti-sense RNA stabilization induces transcriptional gene silencing via histone

deacetylation in S. cerevisiae. Cell. 2007, 131(4):706-17. 177

5 ABBREVIATIONS 201

6 REFERENCES 203

7 CURRICULUM VITAE 222

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1 Introduction

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The eukaryotic cell is the fundamental unit of life of higher organisms. It is characterized by a nuclear compartment, which functions to protect genomic integrity and ensure accurate DNA replication. This compartmentalization provides opportunities for the fine-tuned regulation of both gene expression and RNA processing that are not available in prokaryotes. However, it also means that eukaryotic cells require a machinery that is able to transport thousands of proteins and RNAs in and out of the nucleus so that they can fulfill their appointed role in the life of the cell. Factors implicated in transcription regulation, DNA repair and replication have to be imported into the nucleus, whereas RNAs produced in the nucleus have to be exported, either to accomplish their function in protein synthesis or to mature into functional particles. In particular, cell fate is fundamentally coupled to the faithful expression of its genes, which regulate proliferation, signalling, cell cycle and death. The central process of efficient eukaryotic gene expression hinges on the ability of messenger RNA (mRNA) to transfer the genetic information from the nuclear genome to the cytoplasm, where it can be translated into proteins. Export of mRNAs is therefore indispensable for normal function of a cell.

The following introductory chapters will briefly run through studies that have led to the identification of the cellular factors mediating nucleocytoplasmic transport and describe in detail the complex lives of eukaryotic mRNAs, from their transcription to their export through nuclear pores.

1.1 Nucleocytoplasmic transport: an overview

Embedded in the nuclear envelope are the nuclear pore complexes (NPCs) that form aqueous channels protruding into both the nuclear and cytoplasmic compartments. As the unique site for nuclear entry and exit, these large proteinaceous structures are the gatekeepers for nucleocytoplasmic trafficking. They have evolutionarily conserved function and architecture (Cronshaw et al., 2002; Macara, 2001; Stoffler et al., 1999;

Yang et al., 1998) although their calculated mass is of 60 MDa in yeast and 120 MDa in vertebrates (Cronshaw et al., 2002; Miller and Forbes, 2000; Rout et al., 2000). High-

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resolution electron microscopic studies reveal that NPCs display an eightfold ring- shaped radial symmetric structure composed of a central core, a nuclear basket that extends into the nucleoplasm, and a series of filamentous extensions that reach into the cytoplasm (Figure 1). These cytoplasmic fibrils and nuclear basket are the initial docking sites for import and export complexes, respectively, before they move through the central gated channel (reviewed in Fabre and Hurt, 1997; Wente, 2000).

Figure 1. The arrangement of nuclear pore complexes in the nuclear envelope. (A) Schematic diagram of the nuclear pore complex (NPC). (B) A scanning electron micrograph of the nuclear side of the nuclear envelope of an oocyte. (C) Thin section electron micrograph, showing a side view of two nuclear pore complexes (brackets). (D) Electron micrograph showing face-on views of negatively stained nuclear pore complexes from which the membrane has been removed by detergent extraction. (From Molecular Biology of the Cell 4^th edition).

Proteomic characterization of the mammalian and yeast NPC has shown that it comprises multiple copies of about 30 distinct proteins, called nucleoporins distributed either symmetrically or asymmetrically between the nuclear and cytoplasmic faces of the pore (reviewed in Allen et al., 2000; Rout et al., 2000;

Stoffler et al., 1999). Importantly, half of these nucleoporins contains conserved

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domains rich in phenylalanine-glycine (FG) repeats, which provide primary docking sites for nucleocytoplasmic transport factors as they transverse the NPC (see also sections 1.2 and 1.3.1; Buss et al., 1994; Katahira et al., 1999; Kehlenbach et al., 1999; Radu et al., 1995). Movement through the pores occurs freely for ions and small molecules up to a size of approximately 40 kDa, but those of larger molecules, such as RNAs and proteins, are carefully controlled and require active transport mediated by carrier proteins, termed nuclear transport receptors. These latter proteins are defined by at least three common features: they bind a cargo, shuttle between the nucleus and cytoplasm and directly interact with FG-nucleoporins.

Studies from the past decades revealed that the vast majority of nucleocytoplasmic transport, including proteins and most classes of RNAs (transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and microRNA (miRNA)), is mediated by a superfamily of transport receptors known as karyopherins (or importin-β family members) (reviewed in Chook and Blobel, 2001; Gorlich, 1998; Gorlich and Kutay, 1999). This superfamily comprises 14 members in yeast and more than 20 in higher eukaryotes. Karyopherins mediate either nuclear import (also called importins) or nuclear export (also called exportins) by interacting directly or via adaptor proteins with their cargoes and nucleoporins. These proteins associate with specific sequences on their cargoes, genuine shipping documents, that provide selectivity to the transport process.

Cargoes destined for the nucleus carry a nuclear localization signal (NLS), whereas substrates to be exported from the nucleus harbor a nuclear export sequence (NES). A common feature of karyopherins is their binding to the small GTPase Ran, which regulates their association with cargoes and controls the directionality of nuclear transport (Figure 2).

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Figure 2. Scheme of import (left) and export (right) of proteins. In the cytoplasm an importin binds to cargo molecules containing a NLS and mediate interactions with the NPC to translocate the complex into the nucleus. Nuclear RanGTP binds to the importin and induces cargo release from the complex. The importin-RanGTP complex is then recycled to the cytoplasm. In the case of export, nuclear cargo with a NES binds to an exportin associated with RanGTP and the trimeric complex is then translocated through the NPC to the cytoplasm, where RanGTP is removed from the complex by GTP hydrolysis and the exportin dissociates from the cargo and is recycled back to the nucleus (adapted from Pemberton and Paschal, 2005).

Karyopherins are therefore central to much of the traffic through the NPC. Strikingly, unlike other RNAs, nuclear export of the majority of cellular mRNAs relies on a distinct subset of shuttling factors that are not structurally related to karyopherins and does not directly depend on the RanGTP-RanGDP gradient.

1.2 mRNA: packing and delivery of a genetic message

In eukaryotes, the export of mRNAs from the nucleus to the cytoplasm appears to be a much more complicated process than protein and other RNAs export. First, the expression of a protein-coding gene involves several steps, which are: transcription of the gene generating a precursor messenger RNA (pre-mRNA); extensive processing of this

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pre-mRNA (section 1.2.1) and export from the nucleus to the cytoplasm of the correctly processed “mature” mRNA (section 1.2.2) for it subsequent translation into protein. A picture emerges of tight interconnections between processes controlling mRNA biogenesis and mRNA export involving multiple protein complexes (section 1.2.3). This extensive coupling is likely to function as a quality control mechanism (section 1.2.4) by ensuring that only fully mature mRNAs reach the cytoplasm (Figure 3).

Figure 3. Simplified view of eukaryotic gene expression. The nuclear mRNA processing steps of 5′-end capping, splicing, mRNA assembly into an mRNP particle, 3′- end cleavage and polyadenylation, mRNP surveillance and RNA export are all coupled processes. Pol II, RNA polymerase II (adapted from Aguilera, 2005).

1.2.1 Biogenesis of eukaryotic mRNA transcripts: birth of messenger ribonucleoproteins

The transcription of eukaryotic genes is carried out by RNA Polymerase II (RNA Pol II), which gives rise to pre-messenger RNAs (pre-mRNAs) (reviewed in Maniatis and Reed, 2002; Moore, 2005; Orphanides and Reinberg, 2002). Upon transcription and throughout their lifetimes, pre-mRNAs are bound to numerous distinct factors, forming

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pre-mRNA ribonucleoprotein complexes (pre-mRNPs). These proteins profoundly influence pre-mRNA processing as well as export, localization, translation and stability of mRNAs. The complexes they form with mRNAs and their precursors are specific and dynamic. Some RNP proteins are subject to dynamic exchange immediately before translocation through the NPC, while others remain stably bound to the resulting mRNAs all the way to ribosomes, before being recycled back into the nucleus by karyopherins (reviewed in Dreyfuss et al., 2002). These factors include the nucleocytoplasmic shuttling hnRNP (heterogeneous nuclear RNP), SR (serine/arginine rich) proteins as well as the EJC (exon-junction complex) and mRNA export factors (Das et al., 2007; Dreyfuss et al., 2002; Gatfield and Izaurralde, 2002; Hacker and Krebber, 2004; Huang and Steitz, 2005;

Hurt et al., 2004; Kataoka and Dreyfuss, 2004; Le Hir et al., 2000a; Le Hir et al., 2000b;

Merz et al., 2007; Shibuya et al., 2004; Singh et al., 2006). Both hnRNP and SR proteins recognize short consensus sequences through their RNA binding domain (reviewed in Dreyfuss et al., 2002), while the EJC is deposited onto spliced mRNAs by the process of pre-mRNA splicing (see below). Before being exportable, a pre-mRNP undergoes an intricate maturation process, including typically several interdependent steps: 5’-capping (1), splicing of intron-containing mRNAs (2), 3’-end cleavage and polyadenylation (3) (reviewed in Proudfoot and O'Sullivan, 2002; Zenklusen and Stutz, 2001).

1) 5’ capping

When pre-mRNA reaches a length of 22-25 nucleotides, it is capped by methyl- guanylation at its 5’ end with concomitant co-transcriptional recruitment of the nuclear cap binding protein complex CBC (composed of Cbp20 and Cbp80). This modification is critical for protection from 5’ exonucleases as well as subsequent steps of splicing, decapping, translation and nuclear mRNA decay (Das et al., 2003). In the cytoplasm, the CBC is exchanged by eIF4E, which participates in mRNA translation initiation (reviewed in Neugebauer, 2002; Proudfoot et al., 2002).

2) Splicing of intronic sequences

Most eukaryotic genes are transcribed into pre-mRNA products containing protein- encoding portions (exons) interspersed with non-coding sequences (introns). Pre-mRNAs can be spliced in several different ways, allowing a single gene to encode multiple

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proteins. The process of splicing is carried out in the nucleus by an RNA-protein complex, called the spliceosome, which promotes the enzymatic removal of non-coding mRNA sequences (introns) and the ligation of corresponding exons (reviewed in Neugebauer, 2002; Proudfoot et al., 2002). In higher eukaryotes, the splicing reaction leads to the deposition of a complex of proteins referred to as the exon-junction complex (EJC), onto mRNPs. The EJC is implicated in post-splicing events such as mRNA nuclear export, nonsense-mediated mRNA decay (a quality-control mechanism that selectively degrades mRNAs harboring premature termination codons) and is thought to enhance the cytoplasmic translation rates by enhancing mRNA stability and recruiting the mRNP to polysomes in the cytoplasm (Nott et al., 2004; Wiegand et al., 2003).

Importantly, the EJC complex is also required for proper localization of a specific mRNA during oogenesis, a process critical for posterior formation in Drosophila development (Le Hir et al., 2001; Vanzo and Ephrussi, 2002). Furthermore, the EJC was reported to regulate germline stem cell differentiation and oocyte specification in Drosophila (Parma et al., 2007) as well as late embryogenesis and proper germline sexual differentiation in Caenorhabditis elegans (C. elegans) (Kawano et al., 2004).

3) 3’-end cleavage and polyadenylation

Upon transcription termination, the nascent transcript is cleaved at its 3’-end, released from the site of transcription and stabilized by the addition of a poly-adenine (poly(A)⁺) tail. Throughout eukaryotes, the whole process takes place in a large complex (500-1000 kDa) that includes poly(A)⁺ polymerase (PAP) and many additional factors. The poly(A)⁺ tail addition occurs on almost all eukaryotic mRNAs and is important for transcription termination, mRNA export (see section 1.3.3) as well as in enhancing translation initiation and determining mRNA stability (reviewed in Maniatis and Reed, 2002;

Neugebauer, 2002; Proudfoot et al., 2002). Therefore, control of poly(A)⁺ tail synthesis could possibly be a key regulatory step in gene expression.

The successful maturation of pre-mRNPs generates large export-competent messenger ribonucleoproteins (mRNPs) endowed with nuclear export and cytoplasmic translation capabilities (reviewed in Dreyfuss et al., 2002).

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1.2.2 Nuclear export of mRNA

The nuclear exit of mRNAs is a complex process (see following sections) and increasing evidence points to the coordination of several classes of proteins and complexes to guide an mRNA along its path from transcription to translation. Most of these factors were identified in screening for conditional yeast mutants that accumulate poly(A)⁺ RNA in their nucleus at the restrictive temperature as well as through genetic and biochemical approaches in yeast, mammalian, and viral systems (reviewed in Nakielny and Dreyfuss, 1999; Stutz and Rosbash, 1998). As stated above, cellular mRNAs exist in dynamic association with multiple distinct proteins and are not exported to the cytoplasm as naked nucleic acids, but rather as mRNPs. It is generally agreed that the export machinery recognizes signals within the proteins of these complexes rather than the mRNA itself. The mRNA export machinery includes numerous mRNA binding proteins, ATPase/RNA helicases, and NPC-associated proteins. Most are essential, and yeast strains carrying conditional mutations in any of these genes show rapid and strong defects in mRNA export under non-permissive conditions.

1.2.2.1 Mex67/TAP an export receptor for most mRNAs

Although it is likely that some mRNA species are exported through alternative pathways, works in different model systems have provided convincing evidence that nuclear exit of bulk mRNAs is specifically dependent on the essential shuttling export receptor Mex67 in yeast or its ortholog TAP/NXF1 in metazoans. Mex67 and TAP fulfill the main features expected for mRNA export receptors. They crosslink to poly(A)⁺ RNA in vivo (Katahira et al., 1999; Segref et al., 1997), they directly interact with FG- nucleoporins at the NPC (Bachi et al., 2000; Katahira et al., 1999; Santos-Rosa et al., 1998; Schmitt and Gerace, 2001; Segref et al., 1997; Strawn et al., 2001), undergo nucleocytoplasmic shuttling (Bachi et al., 2000; Bear et al., 1999; Braun et al., 1999;

Kang and Cullen, 1999; Katahira et al., 1999; Schmitt and Gerace, 2001), and are directly required for mRNA export (Hurt et al., 2000; Santos-Rosa et al., 1998; Segref et al., 1997; Strasser et al., 2000; Zenklusen et al., 2001). Consistent with a direct role in mRNA export, thermosensitive (ts) alleles of the budding yeast MEX67 gene exhibit an

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extremely rapid (within the 5 min range) and strong inhibition of mRNA export after a shift to the restrictive temperature, suggesting that Mex67 is at the heart of the mRNA export process (Segref et al., 1997). TAP, the human homologue of Mex67 (Katahira et al., 1999; Segref et al., 1997), was initially identified as the cellular factor responsible for viral replication by exporting viral genomic transcripts through direct binding to their constitutive transport element (CTE) (Gruter et al., 1998). A role for TAP in cellular mRNA export was first revealed in Xenopus oocyte injection experiments, in which CTE RNA competed in a TAP-dependent manner with mRNA export (Gruter et al., 1998;

Pasquinelli et al., 1997). Direct evidence for a role of Mex67/TAP in mRNA export has been obtained later on in Schizosaccharomyces pombe (S. pombe) (Yoon et al., 2000), C.

elegans (Tan et al., 2000), Drosophila melanogaster (Herold et al., 2001; Korey et al., 2001; Wilkie et al., 2001) and mammalian cells (Braun et al., 2001; Guzik et al., 2001;

Kang and Cullen, 1999). These data together with the report that human TAP can functionally replace Mex67 in yeast (Katahira et al., 1999) indicate that the mRNA export pathway is conserved throughout evolution.

More specifically, Mex67/TAP belongs to the evolutionary conserved family of nuclear export factor (NXF) proteins (reviewed in Izaurralde, 2002), which domain organization has been analyzed in detail (Figure 4).

Figure 4. Domain organization and binding partners of Mex67. From the N to the C- terminus there is a leucine-rich region (LRRs), an NTF2-like domain and a ubiquitin associated (UBA) domain. Yra1 and Yra2 associate with the N-terminal half of Mex67, whereas the binding to Mtr2 and NPC is mediated by Mex67 C-terminal part. Amino acid positions are indicated on the top.

Mex67/TAP can be divided in two main functional parts: an N-terminal (amino acids 1-372) cargo-binding domain and a C-terminal half (amino acids 371-619) essential for Mex67/TAP shuttling and NPC binding (Bachi et al., 2000; Braun et al., 2001; Braun et

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al., 2002; Fribourg et al., 2001; Grant et al., 2002; Levesque et al., 2001; Suyama et al., 2000; Wiegand et al., 2002). Notably, the latter activities have proven to be mutationally inseparable (Kang et al., 2000), thus strongly arguing that the ability of Mex67/TAP to exit the nucleus, and hence to export mRNAs, is mediated by a direct interaction of Mex67/TAP with FG-nucleoporins.

TAP and Mex67 form heterodimers with small proteins, p15 and Mtr2, respectively (Katahira et al., 1999; Santos-Rosa et al., 1998; Suyama et al., 2000), which help dock the complex to NPC and are also essential for mRNA export (Herold et al., 2001;

Wiegand et al., 2002). p15 interacts with the NTF2-like domain of TAP and is proposed to contribute to the proper folding of the C-terminal part of TAP, thereby enhancing the ability of TAP to interact with FG-nucleoporins of the NPC. Accordingly, Mtr2 binds the corresponding domain in Mex67 and is required for Mex67 to associate with the NPC (Santos-Rosa et al., 1998). Despite the lack of sequence similarity between Mtr2 and p15, co-expression of p15 with TAP is required for TAP to functionally replace Mex67 in yeast (Katahira et al., 1999), suggesting that Mtr2 and p15 are functionally equivalent. A model for translocation of mRNPs through the NPC has arisen from these findings proposing that Mex67/TAP-Mtr2/p15 drive mRNPs through the pore by transient and sequential interactions with FG-nucleoporins. Directionality of mRNA transport may be due to the early sequestration of mRNPs on the ribosomes in the cytoplasm, depleting the pool of free (non-translation-engaged) mRNAs in this compartment. The ATPase/RNA helicase Dbp5 located on the cytoplasmic side of the NPC may also contribute to this directionality by promoting the release of mRNPs from the NPC (see section 1.2.2.3).

1.2.2.2 Recruiting factors for Mex67/TAP on transcripts

Concurrent with resolving Mex67-mediated mRNP translocation through the NPC, much effort has been put into dissecting the pathway(s) leading to Mex67 recruitment on mRNPs. It was originally reported that Mex67 binds directly to RNA (Segref et al., 1997), however subsequent studies revealed that this interaction is weak implying that Mex67 binding to mRNAs requires adaptor protein(s).

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1.2.2.2.1 Yra1, a coupling protein

To date, the best-characterized adaptor for Mex67/TAP is the essential Yra1 protein in yeast or REF in higher eukaryotes. Yra1/REF was even thought until recently as to be the only adaptor for Mex67/TAP. Yra1/REF was first identified as an RNA-RNA annealing protein (Portman et al., 1997), and was subsequently found to associate directly with mRNA, as shown by gel-shift or UV-crosslinking analyses (Strasser and Hurt, 2000;

Stutz et al., 2000; Zenklusen et al., 2001). Further studies reported that mRNA export is blocked in yra1 mutant cells or concurrently with a depletion of Yra1 protein supporting a direct role in mRNA export for Yra1/REF (Strasser and Hurt, 2000; Stutz et al., 2000;

Zenklusen et al., 2001). Yra1/REF was also found associated with transcriptionally active genes (Lei et al., 2001; Zenklusen et al., 2002), indicating an early recruitment of this mRNA export factor on the growing mRNPs.

A connection between Yra1/REF and Mex67/TAP was first highlighted in budding yeast when Yra1 was uncovered in a screen for proteins that interact genetically with Mex67 (Strasser and Hurt, 2000). Additional studies revealed a direct physical association between Yra1 and Mex67 both in vivo and in vitro (Strasser and Hurt, 2000;

Stutz et al., 2000; Zenklusen et al., 2001). Interestingly, only sub-stoichiometric amounts of Mex67 were found associated with Yra1 (Strasser and Hurt, 2000) and (this study), suggesting that the interaction between Yra1 and Mex67 during transport is transient or weak. The involvement of Yra1/REF in mRNA export and its direct association with both Mex67/TAP and mRNAs led to the proposal that Yra1/REF facilitates the recruitment of Mex67/TAP on mature mRNPs and hence promotes their nuclear exit (Strasser and Hurt, 2000; Stutz et al., 2000; Zenklusen et al., 2001; Zhou et al., 2000). Consistent with this view, strains expressing yra1 mutant proteins, that inefficiently interact with Mex67 in vitro, have significantly lower amounts of Mex67 protein associated with their mRNPs and display an mRNA export defect (Zenklusen et al., 2001). Intriguingly, Yra1 does apparently not shuttle between the nucleus and the cytoplasm in yeast (Strasser and Hurt, 2000; Stutz et al., 2000), indicating that it may be removed from mRNPs before their nuclear exit.

Yra1 belongs to the evolutionarily conserved REF (RNA and Export Factor binding) family, which has several members in most species (Stutz et al., 2000). In S. cerevisiae, Yra1 together with its paralogue Yra2, are the two members of the REF family identified

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so far. Yra2 is not essential for growth and no obvious phenotype is observed in its absence (Zenklusen et al., 2001). However, Yra2 also interacts with Mex67 and can bypass the requirement for Yra1 when over-expressed (Zenklusen et al., 2001), indicating that Yra1 and Yra2 have redundant or overlapping functions. As shown in Figure 5, REF proteins are characterized by a conserved central RNP-motif RNA binding domain (RBD), flanked by Arg-Gly rich (RGG) regions of variable length (N-var and C-var) and short conserved N- and C-termini, referred to as the N- and C-terminal boxes respectively (Rodrigues et al., 2001; Stutz et al., 2000).

Figure 5. Domain organization and binding partners of Yra1. N- and C-terminal regions are highly conserved as well as the central RBD domain. The variable regions N- var and C-var are less conserved between species and within REF members of the same organism. Mex67, Sub2 and RNA binding is mediated by the N- and C-terminal regions of Yra1 (Rodrigues et al., 2001; Strasser and Hurt, 2001; Zenklusen et al., 2001). Amino acid positions are indicated on the top.

Human Yra1/REF, also known as Aly, was first discovered as a cofactor of two transcription factors (LEF-1 and AML) (Bruhn et al., 1997) and soon after as a protein chaperone designated BEF, which was shown to increase transcriptional activation (Virbasius et al., 1999). Thus, Yra1/REF may participate in multiple steps of mRNA biogenesis including transcription and export. Subsequently, murine Aly/REF was shown to interact with TAP and Mex67, and to complement the non-viable YRA1 null mutant (Strasser and Hurt, 2000), indicating functional conservation among REF proteins.

Further evidence for a direct participation of REFs in mRNA export came from experiments in Xenopus oocytes, where injection of recombinant Aly/REF stimulated mRNA export and anti-REF antibodies inhibited export (Rodrigues et al., 2001; Zhou et al., 2000).

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Biochemical studies have shown that REF proteins interact with both Mex67/TAP and RNA through their N- and C-terminal boxes and variable regions, but not through the RBD domain, leaving the central core of REF proteins without any clear function (Rodrigues et al., 2001; Strasser and Hurt, 2001; Zenklusen et al., 2001).

1.2.2.2.1.1 Sub2/UAP56, a recruiting factor for Yra1/REF

Thus, Yra1/REF mediates mRNA export through interaction with the mRNA export receptor, Mex67/TAP, which in turn interacts with the NPC and allows the translocation of mRNPs to the cytoplasm. How then is Yra1/REF recruited on mRNAs? This specific issue was addressed by the Reed and Hurt labs. The former uncovered UAP56, an essential human splicing factor member of an evolutionarily conserved family of DEAD box ATPase/RNA helicases, as an interacting partner of Yra1/REF (Luo et al., 2001).

Using injection experiments in Xenopus oocytes, they then found that excess of UAP56 blocks the export of spliced mRNAs by sequestering Yra1/REF, preventing it from binding to mRNA (Luo et al., 2001). In the meantime, the Hurt lab uncovered that Sub2, the yeast counterpart of human UAP56, interacts both genetically and physically with Yra1/REF in yeast and importantly, determined that the binding of Sub2 and Mex67 to Yra1 are mutually exclusive (Strasser and Hurt, 2001). In addition, this and other labs found that Sub2/UAP56, a bona fide splicing factor (Kistler and Guthrie, 2001; Libri et al., 2001; Zhang and Green, 2001), is a general mRNA export factor for transcripts derived from both intron and non-intron containing genes (Gatfield et al., 2001; Jensen et al., 2001a; Luo et al., 2001; Strasser and Hurt, 2001). Together these data defined a more detailed picture of the mRNA export pathway where Sub2/UAP56 recruits Yra1/REF to nascent mRNPs early during transcription and is then displaced by Mex67/TAP, which targets the mRNPs to the NPC. UAP56 has also been visualized at transcription sites in Chironomus and human cells (Custodio et al., 2004; Kiesler et al., 2002), suggesting that the Yra1/REF co-transcriptional recruitment by Sub2/UAP56 might be conserved.

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1.2.2.2.1.2 TREX, a complex coupling transcription to export

Yeast Yra1 and Sub2 interact genetically and physically with components of the so- called THO complex (consisting of Hpr1, Mft1, Thp2, and Tho2), known to function in transcription elongation (Chavez et al., 2000; Piruat and Aguilera, 1998). Likewise, REF and UAP56 have been found to interact with the human counterparts of the complex (Strasser et al., 2002). Importantly, deletion of individual THO components results in nuclear mRNA export defects (Libri et al., 2002; Schneiter et al., 1999; Strasser et al., 2002), providing a functional link between transcription and mRNA export. In light of these observations, this THO-Sub2-Yra1 multiprotein complex was subsequently termed the TREX complex, proposed to couple transcription and export (Strasser et al., 2002).

Since the co-transcriptional recruitment of Sub2 to active genes is Hpr1-dependent (Zenklusen et al., 2002), the THO complex was proposed to facilitate loading of Sub2 and Yra1 onto the nascent mRNA. However, this model is under debate since an ensuing study found that Sub2 is clearly bound to nascent mRNAs, whereas Yra1 recruitment to genes is partially RNA-independent (Abruzzi et al., 2004). These data suggest that Yra1 is first recruited to the site of transcription through interaction with the transcription machinery and/or the THO complex, whereas Sub2 is recruited on emerging mRNAs.

Consistent with this view, Reed and colleagues recently found that human Aly/REF binds closest to the 5’ end of spliced mRNAs and associates with the cap binding protein Cbp80 (Cheng et al., 2006). However, unlike the THO-dependent co-transcriptional recruitment of Yra1/REF and Sub2/UAP56 in yeast, the authors found that human TREX is recruited to mRNAs via Aly/REF and Cbp80. This apparent discrepancy between studies in yeast and human could be reconciled if one considers the fact that yeast TREX is recruited co-transcriptionally (Abruzzi et al., 2004; Strasser et al., 2002; Zenklusen et al., 2002), while human TREX is loaded onto mRNAs during splicing (see section 1.3.2 and Masuda et al., 2005).

A growing body of evidence supports a model in which the TREX complex would contribute to co-transcriptional mRNP assembly and ensure packaging of the mRNA into an exportable mRNP complex (Abruzzi et al., 2004; Libri et al., 2002; Strasser et al., 2002; Zenklusen et al., 2002). Both the RNA annealing activity of Yra1/REF and the putative ATPase activity of Sub2/UAP56 might facilitate the correct folding of mRNP complexes, and the THO complex might ensure efficient recruitment of these two

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proteins to nascent transcripts (Huertas and Aguilera, 2003; Strasser et al., 2002). Later, the binding of Mex67/TAP-Mtr2/p15 to Yra1/REF competes and displaces Sub2/UAP56 from the mRNPs (Strasser and Hurt, 2001), which are finally translocated through the NPCs by direct interaction of Mex67/TAP-Mtr2/p15 with FG-nucleoporins (Figure 6).

Figure 6. Model for Mex67 recruitment on mRNPs via the TREX complexes in yeast. During early transcription, the tetrameric THO complex, consisting of Hpr1, Tho2, Mft1, and Thp2, becomes associated with the elongating RNA Pol II complex. Following transcription, Hpr1 recruits Sub2 on nascent transcripts, which in turn promotes Yra1 recruitment. Binding of Mex67 is thought to displace Sub2 on Yra1 and induces subsequent export of mRNAs by interacting with nucleoporins lining the pore. In contrast to its mammalian homologue, Yra1 has not yet been shown to shuttle, suggesting a release of Yra1 from the mRNPs just before their translocation through the NPC.

1.2.2.2.2 Other recently identified adaptors for Mex67/TAP

This unique linear pathway for mRNA export implies that the nuclear export machinery, defined minimally by Yra1/REF and Mex67/TAP, does not distinguish among the thousands of mRNAs. What if, on the other hand, mRNAs were exported

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selectively as groups by different RNA-binding proteins? This latter suspicion would entail that Yra1/REF is not the only protein chaperoning the association of Mex67/TAP with mRNPs. Consistently, recent studies in budding yeast have shown that Yra1 does not associate with all transcripts and some mutations in Yra1 that abolish binding to Mex67 in vitro reduce, but do not eliminate, association of Mex67 with mRNPs (Hieronymus and Silver, 2003; Lei et al., 2001; Lei and Silver, 2002; Zenklusen et al., 2001). This and the recent discovery that unlike Mex67/TAP and Sub2/UAP56, Yra1/REF is not essential for mRNA export in Drosophila and C. elegans (Gatfield and Izaurralde, 2002; Gatfield et al., 2001; Herold et al., 2003; Longman et al., 2003;

MacMorris et al., 2003), led to the speculation that Mex67 and its metazoan homolog might have other evolutionarily conserved adaptor(s) that mediate their association with mRNP and consequently that all mRNAs are not exported through a single linear pathway. Given the vast complexity of the mRNA export pool, it is not so surprising that alternative pathways to the TREX/Mex67 pathway might exist.

Accordingly, Steitz and co-workers presented evidence that the SR splicing factors RNPS1 (Huang et al., 2003), 9G8 and SF2 (Huang et al., 2004) promote the recruitment of TAP to metazoan mRNPs. Interestingly, 9G8 and SF2 do so only when hypo- phosphorylated (Huang and Steitz, 2005; Huang et al., 2004; Lai and Tarn, 2004). Since the phosphorylation state of SR proteins is regulated during splicing (reviewed in Graveley, 2000), the dephosphorylation of SR proteins leading to TAP recruitment on mRNPs could be a mechanism for the selective export of spliced mRNA versus unspliced pre-mRNA. Furthermore, a recent study by the Guthrie lab demonstrates that the closest relative to SR proteins in S. cerevisiae, Npl3, is another adaptor for Mex67 (Gilbert and Guthrie, 2004). Npl3 is the most abundant RNA binding protein (Wilson et al., 1994), which shuttles between the nucleus and the cytoplasm in association with mRNAs (Flach et al., 1994; Lee et al., 1996), and is essential for mRNA export (Lee et al., 1996;

Singleton et al., 1995). In their report, Gilbert and Guthrie show that Npl3 directly interacts with Mex67 and participates in its recruitment to the mRNP (Gilbert and Guthrie, 2004). Interestingly, the recognition of Mex67 by Npl3 also depends on Npl3 dephosphorylation and appears to control Mex67 dependent mRNA export (see also section 1.3.5; Gilbert and Guthrie, 2004).

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1.2.2.3 Additional factors implicated in mRNA export

Apart from Mex67/TAP and the different factors directly involved in its recruitment, several other proteins have a primary active and essential role in mRNA export. In yeast, these proteins include nucleoporins, mRNA binding proteins (Nab2), and proteins that bind the NPC (Gle2, Gle1, and Dbp5). All of them have metazoan counterparts.

A worthy of note player in mRNA export is Nab2, an essential shuttling RNA- binding protein that is one of the major proteins associated with nuclear poly(A)⁺ RNA in vivo (Anderson et al., 1993). Nab2 interacts with Yra1 (Kashyap et al., 2005) and is required for both poly(A)⁺ tail length control and nuclear export of mRNA (Anderson et al., 1993; Green et al., 2002; Hector et al., 2002), providing an important link between the termination of mRNA polyadenylation and nuclear export. Notably, a recent study shows that the ubiquitin E3 ligase Tom1 is required for export of Nab2 bound mRNPs but does not affect the export of mRNPs associated with Npl3 (Duncan et al., 2000), raising the enticing hypothesis that Nab2 and Npl3 represent different export pathways devoted to specific classes of transcripts (see section 1.3.5).

Another actor in the mRNA export pathway is the nonessential protein, termed Gle2, which directly associates with the FG-nucleoporin Nup116. Mutations in Gle2 and its S.

pombe homolog, Rae1, induce a rapid block in mRNA export (Bailer et al., 1998; Brown et al., 1995; Murphy and Wente, 1996). Importantly, human Rae1 (hRae1) can complement the export deficiency of Rae1 in S. pombe, again implying strong evolutionary conservation (Bharathi et al., 1997). Yeast Gle2 is normally associated with the NPC (Murphy and Wente, 1996), but studies have demonstrated that hRae1, while predominantly NPC associated, is also a shuttling protein that can associate with poly(A)⁺ RNA in vivo (Kraemer and Blobel, 1997; Pritchard et al., 1999). Evidence has been presented that Gle2 and Mex67 directly interact in S. cerevisiae (Zenklusen et al., 2001), in S. pombe (Yoon et al., 2000), and in vitro binding assays suggested a direct interaction between hRae1 and the C-terminal half of TAP (Bachi et al., 2000). In S.

pombe, Gle2/Rae1, but not Mex67, is essential for mRNA export (Yoon et al., 2000).

This contrasts with S. cerevisiae where Mex67, but not Gle2, is essential, suggesting that

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these proteins have overlapping or redundant functions. Overall, the role played by Gle2/Rae1 in nuclear mRNA export is still unclear.

A third interesting protein highly conserved among eukaryotes is the essential Gle1 protein, located on the cytoplasmic side of the NPC and required for mRNA export (Del Priore et al., 1996; Murphy and Wente, 1996). Gle1 was originally identified in yeast as RSS1 in a screen for high copy suppressors of the temperature-sensitive nup159-1 allele (Del Priore et al., 1996), revealing a functional link between Gle1 and the nuclear pore.

Accordingly, Gle1 is anchored to the pore through interactions with the FG-nucleoporin Nup100 and Nup42/Rip1, a non-essential nucleoporin required for export of heat-shock mRNAs (Murphy and Wente, 1996; Strahm et al., 1999). Because mutations in Nup159, Gle1 and Nup42/Rip1 do not result in a general block of all nucleocytoplasmic transport processes, but rather in strong defects in mRNA export, these cytoplasmic fibrils associated proteins appear to have a primary role in mRNA export and are likely to provide essential docking sites, preferentially used by the mRNA export machinery.

Consistent with a role in mRNA export, a synthetic lethal screen with a gle1 ts mutant identified three mutants that displayed specific defects in mRNA export with no apparent perturbation of protein import or export (York et al., 1999). Although Gle1 has not been shown to bind RNA or to shuttle in yeast, its human homologue was recently reported to shuttle between the nucleus and the cytoplasm (Kendirgi et al., 2003) and to be required for mRNA export (Watkins et al., 1998). Importantly, the shuttling domain of hGle1 acts as a dominant-negative export inhibitor of both bulk poly(A)⁺ RNA and specific mRNA transcripts (Kendirgi et al., 2003).

The most is known about Dbp5, an essential DEAD box RNA helicase that is probably involved in the final release of mRNPs from the nuclear pores (see below).

Dbp5 is required for poly(A)⁺ RNA export from the nucleus in both yeast and vertebrates (Schmitt et al., 1999; Snay-Hodge et al., 1998; Tseng et al., 1998) and is normally concentrated at the cytoplasmic filaments of the NPC by interacting with Gle1 and Nup159 (Strahm et al., 1999). Notably, the combination of mutations eliminating both the Nup159 and the Gle1 binding sites for Dbp5 results in synthetic lethality, suggesting that the nuclear rim localization of Dbp5 is critical for its function (reviewed in Cole and

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Scarcelli, 2006). Because the ATP-dependent RNA unwinding activity of Dbp5 and its nuclear pore localization are essential for its biological function, Dbp5 has been proposed to participate in mRNA export by restructuring mRNP complexes during translocation through and release from the NPC, as well as by removing non-shuttling hnRNP proteins from the mRNPs during export (Hodge et al., 1999; Lei et al., 2001; Schmitt et al., 1999;

Snay-Hodge et al., 1998; Strahm et al., 1999). This attractive model could give a clue into how mRNAs are released from export complexes in the cytoplasm, since RanGTP is not involved in mRNA export. Evidence for a significant remodeling of mRNPs throughout nuclear exit came from electron microscopic analysis of the giant (30-45 kb) Balbiani ring (BR) mRNA of the insect Chironomus tentans. The BR transcript coated with proteins is packed into a compact ring-shaped mRNP, but it gradually unfolds as it passes through the NPC and emerges extended on the cytoplasmic side (reviewed in Daneholt, 2001). A more recent study reported that much more Mex67 is present on mRNPs in dbp5 mutant cells compared to wt cells (Lund and Guthrie, 2005), supporting the idea that Dbp5’s function is specifically required to displace Mex67 from mRNPs at the cytoplasmic side of the NPC. Notably, a recent thrilling finding shows that Gle1 and the phosphoinositide IP₆ dramatically stimulate Dbp5’s ATPase activity in vitro (Alcazar-Roman et al., 2006; Weirich et al., 2006), which could explain how Dpb5’s activity is controlled spatially so that disassembly of mRNPs does not occur at inappropriate locations. Gle1 and Nup159 may therefore function by providing a platform to which translocating mRNPs bind and then dissociate through the action of Dbp5.

Together, these findings have reinforced a model whereby Dbp5 functions as a molecular motor while bound to the cytoplasmic pore filaments to remodel and pull the mRNPs into the cytoplasm through the energy delivered by ATP hydrolysis. This local activation of Dbp5 could generate directionality and termination of mRNA export.

Interestingly, immunoelectron microscopy in C. tentans shows that Dbp5 can be found associated with the 5’ end of mRNA at early steps of transcription (Zhao et al., 2002).

There is also evidence that Dbp5 shuttles and associates with Yra1-containing complexes (Hodge et al., 1999; Schmitt et al., 1999; Strawn et al., 2001), suggesting a role in earlier steps of mRNA export. These observations together with the recent reported genetic and biochemical interactions between Dbp5 and the early transcription factor TFIIH (Estruch

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and Cole, 2003) further suggest that Dbp5 might be recruited and functions in early transcription as well.

1.3 mRNA export is coupled to other processes: evidence for an export license

It has become increasingly clear that virtually all steps in gene expression are intimately linked such that splicing, polyadenylation, and capping all affect export (reviewed in Maniatis and Reed, 2002; Proudfoot et al., 2002). It is believed that processing reactions may generate signals on the mRNP necessary for the subsequent interaction with the export machinery, allowing to distinguish immature pre-mRNA from fully processed mRNA. The coupling of mRNA processing and export ensures fidelity and efficiency of gene expression as well as a tight control that prevents incompletely or improperly processed mRNAs to reach the cytoplasm, where they may have deleterious effects (reviewed in Zenklusen and Stutz, 2001).

1.3.1 mRNA export and transcription

Increasing evidence points to a direct coupling between transcription and export. In this view, the CTD (C-terminal domain) of RNA Pol II plays a central role, since it acts as a loading platform to coordinate the recruitment of factors involved in mRNA processing and mRNA export on the nascent mRNAs (Proudfoot, 2000). This might be best exemplified by the mRNA export factor Npl3, which interacts genetically and physically with components of the transcription machinery and is recruited to mRNAs at an early stage of transcription via interaction with the RNA Pol II (Lei et al., 2001). Since Npl3 export requires ongoing transcription, it was thus suggested that Npl3 is exported in association with mRNA (Flach et al., 1994; Lee et al., 1996). Yra1/REF was also shown to associate with nascent mRNAs during its synthesis (see sections 1.2.2.2.1-2; Cheng et al., 2006; Lei et al., 2001; Strasser et al., 2002; Zenklusen et al., 2002) and to bind some

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transcription co-activators in mammalian cells (Bruhn et al., 1997; Virbasius et al., 1999).

Since both factors are essential for mRNA export and act as adaptors for Mex67, these observation highlight that mRNAs are targeted for nuclear export already during transcription.

Finally, recent studies identified additional components of the conserved mRNA export machinery, which tighten the connection between transcription and mRNA export (see also section 1.4; Fischer et al., 2002; Gallardo and Aguilera, 2001; Lei et al., 2003;

Rodriguez-Navarro et al., 2004; Strasser et al., 2002). Sac3, which is localized in the nuclear basket and cytoplasmic fibrils of the NPC, has been shown to interact genetically with Yra1, Sub2 and Mex67 and physically with Mex67 (Fischer et al., 2002; Lei et al., 2003; Strasser et al., 2002). Subsequent work demonstrated that Mex67 accumulates at the nuclear rim in a sac3 mutant background (Lei et al., 2003). These findings suggest that Sac3 may be required to release Mex67 from the mRNP after translocation through the NPC. Importantly, Sac3 also tightly binds Thp1, which was previously implicated in transcription elongation and genome stability (Gallardo and Aguilera, 2001). Moreover, the Hurt lab showed that these two proteins are required for mRNA export (Fischer et al., 2002) and interact with Sus1, a functional component of the SAGA histone acetylase complex involved in transcription activation (Rodriguez-Navarro et al., 2004). This indicates a direct coupling of transcription and export mediated by a physical connection between SAGA and the Thp1–Sac3 complex. In addition, the deletion of Sus1 causes both transcriptional and mRNA export defects (Rodriguez-Navarro et al., 2004), consistent with a role in both processes. The identification of such a supercomplex that couples SAGA-dependent gene expression to mRNA export, led the authors to call it the TREX-2 complex. On the basis of these results, it is possible that the Sac3-Thp1 complex associates with the mRNA during transcription and participates in the docking and subsequent translocation of Mex67-containing mRNPs through the NPC. Metazoan counterparts of Sac3 and Thp1 have been identified, suggesting that this might also hold true in higher eukaryotes.

In summary, the current data suggest that TREX and TREX-2 complexes represent parallel and maybe overlapping pathways linking intranuclear mRNA biogenesis and export (Figure 7). Whether TREX and TREX-2 components bind distinct mRNAs or are sequentially recruited to the same transcripts remains to be determined.

The ubiquitin pathway regulates multiple steps of Mex67-mediated mRNA export

Thesis

Reference

The ubiquitin pathway regulates multiple steps of Mex67-mediated mRNA export

The Ubiquitin Pathway Regulates Multiple Steps of Mex67-mediated mRNA Export

THÈSE

The Ubiquitin Pathway Regulates Multiple Steps of Mex67-mediated mRNA Export

THÈSE

Table of contents

1 Introduction

1.1 Nucleocytoplasmic transport: an overview

1.2 mRNA: packing and delivery of a genetic message

1.2.1 Biogenesis of eukaryotic mRNA transcripts: birth of messenger ribonucleoproteins

1.2.2 Nuclear export of mRNA

1.3 mRNA export is coupled to other processes: evidence for an export license

1.3.1 mRNA export and transcription