Thesis
Reference
Dissecting the functional links between the mRNA export adaptor Yra1, the E3 ubiquitin ligase Slx5-Slx8 and genome stability
INFANTINO, Valentina
Abstract
Yra1 is an essential RNA binding protein in S. cerevisiae. The aim of this thesis was to elucidate the function of Yra1 ubiquitination by the STUbL Slx5-Slx8. We examined whether Yra1 ubiquitination by Slx5-Slx8 leads to proteasomal degradation (Chapter 1). Our studies indicate that Yra1 ubiquitination by Slx5-Slx8 does not promote Yra1 degradation. One hypothesis is that Yra1 ubiquitination by Slx5-Slx8 may be important for Yra1 auto-regulation.
We could not conclude whether Yra1 ubiquitination by Slx5-Slx8 is important for Yra1 splicing auto-regulation (Chapter 2). We analyzed the effect of Yra1 overexpression (OE) mutants on pathways in which Slx5-Slx8 have been implicated. We analyzed the effect of Yra1 overexpression on the DNA Damage Response (Chapter 3) and on irreparable DSBs (Chapter 4), as well as on Chromosome Segregation (Chapter 5). Overall, this study supports the importance of Yra1 regulation in two pivotal pathways: the DNA Damage Response and the Spindle Positioning.
INFANTINO, Valentina. Dissecting the functional links between the mRNA export adaptor Yra1, the E3 ubiquitin ligase Slx5-Slx8 and genome stability. Thèse de doctorat : Univ. Genève, 2017, no. Sc. 5123
DOI : 10.13097/archive-ouverte/unige:112567 URN : urn:nbn:ch:unige-1125671
Available at:
http://archive-ouverte.unige.ch/unige:112567
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Dissecting the functional links between the mRNA export adaptor Yra1, the E3 ubiquitin ligase Slx5-Slx8 and genome stability.
THÈSE
présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Valentina INFANTINO de
COMO (Italie)
Thèse N°5123
GENÈVE unicopy
2017
Acknowledgement
I would like to thank all the people that contributed to the work described in this thesis.
I would like to express my deep gratitude to the Professor Francoise Stutz, my research supervisor, for giving me the opportunity to work in her laboratory and grow as research scientist. She was always demanding the best preparation for academic challenges. She was always stimulating discussions encouraging to share scientific data. She supported me in the attendance of various conferences from the beginning until the end that helped me to develop a strong scientific critical sense crucial for proceeding in this work.
I would like to thank my committee members: the Professor Didier Picard and Thanos Halazonetis for the important discussions and suggestions given for my work during the TAC examinations.
I would like to thank the members of my PhD Thesis jury: the Professors Didier Picard and Vincent Geli for the important points raised during my Thesis Defence.
I wish to acknowledge the Professors Didier Picard and Jean-Claude Martinou for their availability in valuating the final exam that allowed me to proceed with the writing of my Thesis.
I would like to offer my special thanks to the lab mates that supported and helped the work described in this thesis. The work of Evelina Tutucci and Noel Yeh Martin was important for defining the starting point of this project and with them I shared also important aspects of this work along almost two years of my PhD.
Thanks to the work of Ivona Bagdiul I was able to present the complexity of Yra1 Ubiquitination. She also supported the work in other important aspects encouraging also discussions. I wish to acknowledge the help provided by Varinia Garcia Molinero that, even in a short time, gave important contributions to this project.
I would like to express my very great appreciation to the work performed by Audrey Zihlman, my first master student, really good in work organization and efficiency.
I am particularly grateful for the assistance given by Geraldine Silvano for daily laboratory life.
Important aspects in this work were achieved thanks to the support of the collaborators. Thanks to the Dr. Benoit Palancade I learnt how to make scientific
“spaghetti” (and not just pasta) for the preparation of the Mass Spec samples.
Moreover, he was always available for interesting discussions. I was fortunate to have the chance to work for one week with the Dr. Chihiro Horigome in the Susan Gasser laboratory who patiently teached me the Zoning microscopy technique.
I would like to thanks the Dr. Christoph Bauer and Jérome Bosset from the Bioimaging Center for the technical support for the microscopy part of this work.
I am grateful to the NCCR PhD program that allowed me to start my PhD experience with a challenge year characterized by interesting courses and giving the opportunity to rotate in three different laboratories for three months. Thanks to the Professors Francois Karch, Francoise Stutz and Monica Gotta to accept me for three months to work in their laboratory.
I would like to acknowledge the Bicell Department for the nice environment and the high quality seminars organized.
I would like to express my special thanks to the lab mates that with food, laughing, and smiling improved the quality of this work. Thanks to Manuele, Andrea, Mariana, Lorane, Evelina, Noel, Claudia, Deborah, Dario, Nataliia, Jatinder, Julien, Geraldine, Ivona, Varinia, Audrey, Anna and Francoise.
I would like to thank my friends that supported me during these years. I would like to thank our Italian community of Geneva that for strange physic property was formed and was crucial for sustaining this work. Thanks to Claudia, Angela, Erica, Melissa, Andrea, Manu, Sandra, Adriano, Marco, Ale, Valentina, Stefano, Giulia, Antonio, Peppe, Stefano. I would like to thank also my sisters Giada, Simo, Luci, Valeria, Elena, Betty, Sheila and Silvia.
Finally and foremost I would like to thank my Mamma e Papà for the constant support and love they give me and my husband Paolo for reinforcing the motivation, difficult to keep high in hard periods and for remembering me that “everything will be Good”.
Summary
Yra1 is an essential RNA binding protein in S. cerevisiae, described as an mRNA export adaptor mediating the loading of the general mRNA export factor Mex67 on the mRNA ribonucleoprotein particles to allow nuclear mRNA export (Tutucci and Stutz, 2011).
We previously showed that Yra1 ubiquitination by Tom1 is important for mRNA export probably as quality control system to displace Yra1 from fully processed mRNP prior to nuclear exit (Iglesias et al., 2010).
Interestingly, a former PhD student of the laboratory showed that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligase (STUbL) Slx5-Slx8.
Consistently, in collaboration with Benoit Palancade (Institut Jacques Monod, Paris), we found that Yra1 is also SUMOylated by the SUMO E3 ligases Siz1 and Siz2, and de-SUMOylated by the SUMO protease Ulp1. Poly(A)+ RNA localization experiments in the absence of Slx5-Slx8 however indicated no effect in mRNA export. Moreover, the growth and poly(A)+ RNA export defects of the Δtom1 strain were not enhanced in combination with Δslx5 or Δslx8 deletions (E. Tutucci, unpublished data).
In light of these results, the aim of this thesis was to elucidate the function of Yra1 ubiquitination by Slx5-Slx8 in a process possibly distinct from mRNA export.
First of all, since many substrates ubiquitinated by Slx5-Slx8 are targeted to the proteasome (Schweiggert et al., 2016; Sriramachandran and Dohmen, 2014; Xie et al., 2007b; Xie et al., 2010), we examined whether Yra1 ubiquitination by Slx5-Slx8 leads to proteasomal degradation (Chapter 1). Our studies indicate that Yra1 ubiquitination by Slx5-Slx8 does not promote Yra1 degradation and confirm that Yra1 is a very long-lived protein consistent with earlier observations (Christiano et al., 2014). However, Yra1 protein levels have to be tightly regulated since Yra1 overexpression is detrimental to cell growth and causes mRNA export defects (Espinet et al., 1995; Preker et al., 2002; Rodriguez-Navarro et al., 2002).
One hypothesis is that Yra1 ubiquitination by Slx5-Slx8 may be important for Yra1 auto-regulation, since Yra1 was shown to control its own levels by negatively affecting YRA1 pre-mRNA splicing (Dong et al., 2010a; Dong et al., 2007a; Preker
and Guthrie, 2006). Unfortunately, we could not conclude whether Yra1 ubiquitination by Slx5-Slx8 is important for Yra1 splicing auto-regulation (Chapter 2). Although our Mass Spec analyses identified lysine 201 (K15 in our mutants) as the favorable lysine to be Ubiquitinated, the 22 lysines present in Yra1 are highly redundant; it was therefore impossible to generate a minimal yra1KR mutant that abrogates ubiquitination without also presenting mRNA export defects that may indirectly affect splicing. In addition, although Yra1 ubiquitination is abrogated in the
∆slx5/∆tom1 and ∆slx8/∆tom1 double mutants, we could not rely on the splicing auto-inhibition phenotype observed in these strains because of possible indirect effects of other targets on this process. However, we confirmed the importance of the Yra1 highly conserved C-terminal box in splicing auto-inhibition as previously shown (Preker and Guthrie, 2006). These 26 C-terminal amino-acids contain 4 lysines which could potentially be modified and contribute to splicing auto- regulation.
To further investigate the potential role of Yra1 modification by Slx5-Slx8, we analyzed the effect of Yra1 overexpression (OE) mutants on pathways in which this STUbL has been implicated. Slx5-Slx8 was shown to be important in maintaining genome stability (Mullen et al., 2001; Prudden et al., 2007) at different levels. First it was involved in the DNA damage response (DDR) (Burgess et al., 2007), specifically in the anchoring of irreparable Double Strand Breaks (DSBs) to the nuclear pore (Cook et al., 2009; Nagai et al., 2008; Therizols et al., 2006). Second, it was also linked to spindle positioning, spindle elongation and chromosome segregation (Hirota et al., 2014; Schweiggert et al., 2016; van de Pasch et al., 2013). For these reasons, we analyzed the effect of Yra1 overexpression on the DNA Damage Response (Chapter 3) with particular emphasis on irreparable DSBs (Chapter 4), as well as on pathways underlying Chromosome Segregation (Chapter 5).
We observed that yra1 OE mutants are sensitive to genotoxic drugs and genetic interaction analyses with homologous recombination (HR) components reveal synthetic sickness with Δrad52 but not with Δexo1, Δsae2 and Δmre11. Interestingly, the yra1 OE mutants increase the DNA damage sensitivity of the DDR components previously mentioned, suggesting a functional link of Yra1 to DNA lesions repaired by HR. In this regard, the Gasser laboratory has shown that irreparable DSBs are recruited to the Nuclear Pore in a process dependent on Slx5-Slx8 (Horigome et al., 2014; Nagai et al., 2008). We asked whether Yra1 may contribute to this process.
First, we observed that Yra1 is able to relocate a random DNA locus to the nuclear periphery independently of transcription. Furthermore, our data indicate that Yra1 is recruited to an irreparable HO-induced DSB in a transcription-independent manner and after extensive resection. At this point however, it is unclear whether DSB localization depends on Yra1 binding or whether DSB repositioning to the nuclear pore promotes Yra1 association. To address this question, current work aims at correlating the recruitment of wild-type versus mutant Yra1 to a repairable DSB with repair efficiency.
The importance of Yra1 regulation in genome stability is not only related to DDR but is also relevant to Chromosome segregation. Indeed, our experiments show that Yra1 OE mutants present Chromosome instability phenotypes and genetic interactions suggest that proper Yra1 expression contributes to Spindle Positioning through the Kar9 pathway.
Overall, this study supports the importance of Yra1 regulation in two pivotal pathways ensuring genome stability: the DNA Damage Response and the Spindle Positioning. It is currently unclear whether the yra1 OE mutants increase DNA damage or compromise DNA repair. Furthermore, the extent to which the observed phenotypes reflect direct, potentially RNA-independent functions of Yra1, or are merely due to mis-regulation of gene products involved in these processes will require further investigation, and may be questions for which it may be difficult to obtain definitive answers.
Résumé
Yra1 est une protéine de liaison à l’ARN essentielle dans S. cerevisiae, décrite comme un facteur requis pour l’export des ARNs messagers du noyau vers le cytoplasme en agissant comme médiateur de la liaison entre l’ARN et le récepteur d’export Mex67 sur les particules ribonucléoprotéiques (mRNP) (Tutucci and Stutz, 2011).
Nous avons précédemment montré que l'ubiquitination de Yra1 par la E3 ligase Tom1 est importante pour l'export des ARNs messagers, probablement en agissant comme système de contrôle de qualité pour la dissociation de Yra1 du complexe mRNP mature avant l’export du noyau vers le cytoplasme (Iglesias et al., 2010).
Fait intéressant, une ancienne doctorante du laboratoire a montré que Yra1 est aussi modifié par l’ubiquitine ligase SUMO-dépendante (STUbL) Slx5-Slx8.
En collaboration avec le Dr. Benoit Palancade (Institut Jacques Monod,Paris), nous avons montré que Yra1 est également sumoylée par les SUMO E3 ligases Siz1 et Siz2 et désumoylée par la SUMO protéase Ulp1. Les expériences de localisation d’ARN poly(A)+ en absence de Slx5-Slx8 n’ont montré aucun effet sur l’export d’ARN. De plus, les défauts d’export d’ARN poly(A)+ de la souche Δtom1 ne sont pas aggravés en absence de Slx5 et Slx8 (E.Tutucci, données non publiées).
Face à ces résultats, l'objectif de cette thèse était d'élucider le rôle de l’ubiquitination de Yra1 par Slx5-Slx8 dans d’autres processus distincts de l'export des ARNs messagers. Tout d'abord, étant donné que de nombreux substrats ubiquitinés par Slx5-Slx8 ciblent le proteasome (Schweiggert et al., 2016;
Sriramachandran and Dohmen, 2014; Xie et al., 2007; Xie et al., 2010), nous avons examiné si l'ubiquitination de Yra1 par Slx5-Slx8 promeut la dégradation par le protéasome (Chapitre 1). Nos études indiquent que la modification de Yra1 par Slx5- Slx8 ne favorise pas la dégradation de Yra1 et confirment que Yra1 est une protéine très stable (Christiano et al., 2014). Cependant, les niveaux de protéine Yra1 doivent être étroitement contrôlés car la surexpression de Yra1 est préjudiciable à la croissance cellulaire et provoque des défauts d'export d’ARNs messagers dans une fraction des cellules (Espinet et al., 1995; Preker et al., 2002; Rodriguez-Navarro et al., 2002).
Une des hypothèses est que l'ubiquitination de Yra1 par Slx5-Slx8 peut être importante pour l'autorégulation de l’épissage de son propre pré-ARN messager (Dong et al., 2010; Dong et al., 2007; Preker and Guthrie, 2006). Malheureusement, nos expériences n’ont pas permis de conclure si c’était le cas (Chapitre 2). Bien que nos analyses de spectrophotométrie de masse ont identifié la lysine 201 (K15 dans nos mutants) comme la lysine ubiquitinée, les 22 lysines présentes dans Yra1 sont hautement redondantes ; il est donc impossible de générer un mutant yra1KR minimal qui abroge l'ubiquitination sans présenter également des défauts d'export d’ARN messager qui peuvent indirectement affecter l'épissage. En outre, bien que l'ubiquitination de Yra1 soit abrogée dans les doubles mutants ∆slx5/∆tom1 et
∆slx8/∆tom1, nous ne pouvions pas considérer le phénotype d'inhibition de l'épissage observé dans ces souches à cause des effets indirects possibles d’autres cibles sur ce processus. Cependant, nous avons confirmé l'importance de la partie C-terminale hautement conservée de Yra1 dans l'auto-inhibition de l'épissage comme indiqué précédemment (Preker and Guthrie, 2006). A noter que ces 26 acides aminés C- terminaux contiennent 4 lysines qui pourraient être modifiées et contribuer à l'auto- régulation de l'épissage.
Pour comprendre le rôle potentiel de la modification de Yra1 par Slx5-Slx8, nous avons analysé les effets de la surexpression de Yra1 (en utilisant des mutants de surexpression ou yra1 OE), sur les processus dans lesquels cette E3 ligase a été impliquée. L’activité de Slx5-Slx8 a été révélée être importante dans le maintien de la stabilité du génome à différents niveaux (Mullen et al., 2001;Pruden et al., 2007).
D'abord, Slx5-Slx8 a été impliquée dans la réponse cellulaire aux dégâts causés sur l’ADN (DNA Damage Response ou DDR) (Burgess et al., 2007), en particulier dans la relocalisation des cassures double brin (DSB) irréparables au niveau du pore nucléaire (Cook et al., 2009; Nagai et al., 2008; Therizols et al., 2006).
Deuxièmement, Slx5-Slx8 a été lié au positionnement du fuseau mitotique, à son allongement et à la ségrégation des chromosomes (Hirota et al., 2014; Schweiggert et al., 2016;Van de Pasch et al., 2013). Pour ces raisons, nous avons analysé l'effet de la surexpression de Yra1 sur les dommages causés à l'ADN (Chapitre 3), en particulier dans le contexte d’un DSB irréparable (Chapitre 4), ainsi que sur les processus sous- jacents à la ségrégation chromosomique (Chapitre 5).
Nous avons observé que les mutants de surexpression yra1 OE sont sensibles aux drogues génotoxiques et que les analyses d'interactions génétiques avec des
composants de la machinerie de recombinaison homologue (HR) révèlent une interaction synthétique avec ∆rad52 mais pas avec ∆exo1, ∆sae1 et ∆mre11. Fait intéressant, les mutants yra1 OE augmentent la sensibilité des mutants DDR précités aux drogues génotoxiques, ce qui suggère un lien fonctionnel entre Yra1 et l’ADN endommagé réparé par HR. À cet égard, le laboratoire Gasser a montré que des DSB irréparables sont recrutés au pore nucléaire dans un processus dépendant de Slx5-Slx8 (Horigome et al., 2014; Nagai et al., 2008). Nous nous sommes demandés si Yra1 peut contribuer à ce processus. Tout d'abord, nous avons observé que Yra1 peut relocaliser un locus d’ADN aléatoire vers la périphérie du noyau indépendamment de la transcription. De plus, nos données indiquent que Yra1 est recruté sur une cassure double brin irréparable induite par l’endonucléase HO, indépendamment de la transcription et après une résection étendue. À ce stade, il n'est pas clair si la relocalisation du DSB dépend du recrutement de Yra1 ou si le repositionnement du DSB au niveau du pore nucléaire favorise une association avec Yra1. Pour aborder cette question, le travail actuel vise à corréler le niveau de recrutement d’une protéine Yra1 sauvage ou mutante sur un DSB réparable avec l’efficacité du processus de réparation.
L'importance de la régulation de Yra1 dans la stabilité du génome n'est pas seulement liée au DDR mais est probablement aussi pertinente pour la ségrégation des chromosomes. En effet, nos expériences montrent que les mutants yra1 OE présentent des phénotypes chromosomiques instables et les interactions génétiques suggèrent que la surexpression de Yra1 contribue au positionnement du fuseau mitotique en coopération avec Kar9.
Dans l'ensemble, cette étude soutient l'importance de la régulation de Yra1 dans deux voies cruciales assurant la stabilité du génome: la réponse aux dommages causés à l'ADN et le positionnement du fuseau mitotique. Il n’est pas clair à ce point si les mutants yra1 OE augmentent les dommages à l’ADN ou compromettent leur réparation. De plus, il est difficile de définir dans quelle mesure les phénotypes observés reflètent des activités de Yra1 potentiellement indépendantes de sa fonction dans l’export des ARNs, ou s’ils sont dus à des effets indirects liés à des changements d’expression de gènes impliqués dans ces processus. Ces interrogations nécessiteront des investigations plus approfondies, et il pourrait être difficile d’obtenir une réponse définitive à ces questions.
Table of Contents
Introduction 1
Coupling between transcription, processing and mRNA export
through nuclear pores in S. cerevisiae. 2
mRNA 5’ end capping 2
Transcription elongation 3
mRNA 3’ end processing 4
mRNP surveillance and export 5
Gene gating and gene looping at Nuclear Pores 8
The Nuclear Pore Complex 10
Nuclear Organization 12
Nuclear Dynamics during Genome Instability 15
Eroded telomeres tethered at the nuclear envelope 15
Persistent DNA double strand break relocation at the nuclear envelope 16
The mRNA export adaptor Yra1 20
YRA1: the importance to have an intron 24
YRA1 and Genome Instability 26
Project Background 31
Specific aims 35
Results 36
Chapter 1: Yra1 protein half-life 37
Yra1 half-life by blocking translation with Cycloheximide 37
Yra1 protein turnover using metabolic depletion 39
Chapter 2: Yra1 splicing auto-regulation 42
Ubiquitination of Yra1 mutants 42
Yra1 splicing analysis in yra1 mutants 47
Yra1 splicing in ΔE3 Ubiquitin ligase mutants 51
Chapter 3: Yra1 and the DNA damage response pathway 54
Background 54
Sensitivity of Yra1 mutants to DNA damage agents 55
Rad52 foci accumulation in Yra1 mutants 63
Yra1 genetic interactions with DDR components 65
Chapter 4: Yra1 connection to irreparable DSBs and the Nuclear Periphery 77 Yra1 localizes a random locus to the Nuclear Periphery 77 Yra1 localizes a random locus to the nuclear periphery
independently of transcription 80
Yra1 localizes a random locus to the nuclear periphery
under DNA damage induction 80
Yra1 localizes a random locus to the nuclear periphery in Δslx8, Δslx5 and Δsiz2 81
Yra1 is recruited to an irreparable DSB (HO cut) 82
Yra1 binds an irreparable DSB (HO cut) in the absence of transcription 84 Yra1 mutants differently bind an irreparable HO cut 87
Chapter 5: Yra1 and Chromosome Segregation 90
Background 90
Yra1 overexpression causes a Chromosome Segregation defect 91 Yra1 mutant sensitivity to the anti-microtubule drug Benomyl 94 Analysis of spindle positioning and morphologies in HA-Yra1 mutants 96
Yra1 mutants are defective in the Kar9 pathway 102 Strategy to address a potential physical link between Yra1 and the SPB. 103 Chapter 6: Mass Spec Analysis of Yra1 interactors in different conditions 107
Scaffold analysis of Yra1 MS data 113
Chapter 7: Validation of Mass Spec results 118
Yra1 mutants do not show defects in rRNA processing 118 HA-ProtA-Yra1 does not interact with the RPA complex. 120
The ProtA tag (15KDa) affects Yra1 function. 121
Discussion 124
Chapter 1: Yra1 protein half-life 125
Chapter 2: Yra1 splicing auto-regulation 128
Chapter 3: Yra1 and the DNA damage response pathway 131
Chapter 4: Yra1 connection to irreparable DSBs and the Nuclear Periphery 135
Chapter 5: Yra1 and Chromosome Segregation 138
Chapter 6/7: Mass Spectrometry Analysis of Yra1 interacting proteins
in different conditions 141
General discussion 142
Materials and Methods 147
1. Yeast strain constructions 148
1.1 Yeast plasmid shuffling 148
1.2/1.3 Integration of wild-type or mutant HA-YRA1 sequences
into haploid or diploid W303 strains. 148
1.4 Deletion strain constructions 149
2. Media and culture conditions 149
3. Cell cycle arrest and FACS analysis 150
4. Spot test 150
5. Protein extraction and Western blotting 151
6. RNA extraction and quantitative real-time PCR 151
7. Northern blotting 152
8. Chromatin immunoprecipitation 152
9. Ubiquitination and Sumoylation assays 153
10. Purification of His-Yra1 for Mass Spec Analysis of ubiquitinated forms 154 11. Purification of HA-ProtA-Yra1 for Mass Spec Analysis of Yra1 interactors 154
12. Chromosome Segregation assay. 155
13. Zoning assay. 155
14. Rad52 foci analysis. 156
15. Analysis of spindle position and morphologies. 157
16. Mobility assay of LacI-GFP/LacO repeats relative to the SPBs. 157
Supplementary Figures 166
References 171
Introduction
Coupling between transcription, processing and mRNA export through nuclear pores in S. cerevisiae.
Gene expression is ensured by a series of processes coupled to each other that convey the genetic information from the DNA to protein (Crick, 1970). Transcription, RNA processing, mRNA export, translation and degradation co-evolved and are coordinated in space and time. The coupling between different events is mediated by multi-protein complexes that participate in sequential events along the gene expression pathway (Bentley, 2014).
Transcription occurs in the nucleus and involves three different RNA polymerases in S. cerevisiae: RNA Pol I synthesizes the ribosomal precursor RNAs (rRNA), RNA Pol II transcribes protein-coding genes to produce mRNAs, and RNA Pol III is responsible for the synthesis of tRNAs, 5S rRNA and a number of other small RNAs (Turowski, 2013).
The mRNAs synthesized by RNA Pol II have to undergo several processing steps, including capping, splicing and 3’ end processing to form mature messenger ribonucleoprotein particles (mRNPs) competent for export through nuclear pore complexes (NPC) into the cytoplasm (Rondon et al., 2010).
Most mRNA processing steps occur co-transcriptionally and are accompanied by the recruitment of mRNA adapters, such as the RNA binding protein Yra1, the SR like protein Npl3, and the poly(A) binding protein Nab2 that mediate the association of the mRNA export receptor Mex67 with the mRNPs. The ability of Mex67 to interact with mRNA adaptors as well as with nuclear pore components allows to direct mature mRNPs to the NPC for the subsequent proper mRNA export (Tutucci and Stutz, 2011).
mRNA 5’ end capping
The first mRNA processing event is the addition by the capping enzyme of a m7-G-cap at the mRNA 5’ end when the 20-30 first nucleotides have been synthesized (Figure 1A). The m7-G-cap is subsequently bound by the cap-binding complex and protects the mRNA from nuclear and cytoplasmic quality control mechanisms, which recognize non-capped 5’ends leading to 5’!3’ degradation of these transcripts (Das and Das, 2013; Jiao et al., 2010). Co-transcriptional capping is functionally linked to
subsequent events as it ensures optimal transcription elongation, splicing, polyadenylation, mRNA export and translation (Meinel and Strasser, 2015; Topisirovic et al., 2011).
Transcription elongation
Besides 5’capping, pre-mRNA splicing and 3’end processing are also co- transcriptional events. The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a central role in coupling transcription with these maturation steps by mediating the sequential recruitment of the different processing factors according to its phosphorylation state (McCracken et al., 1997b). The CTD is an evolutionary conserved feature specific for RNA PolII (Corden et al., 1985) and consists of a 26 heptad repeats (YS2PTS5PS7) which become differentially phosphorylated along the transcription cycle. At the 5’ end of genes, the CTD is mainly phosphorylated on S5, promoting interaction with the guanine-7-methyltransferase and mRNA capping enzyme, allowing capping to occur (Cho et al., 1997; McCracken et al., 1997a). During elongation, S5 is progressively replaced by S2 phosphorylation favoring the recruitment of the splicing machinery (Morris and Greenleaf, 2000). For instance, the U1 snRNP subunit Prp40 and the SR-like protein Npl3 involved in splicing, interact with phosphorylated CTD suggesting that their co-transcriptional recruitment depends on this interaction (Dermody et al., 2008; Kress et al., 2008).
During elongation, CTD phosphorylation also influences the recruitment of factors implicated in mRNA export, establishing a functional coupling between mRNA transcription and nuclear exit. The transcription and export complex (TREX) consists of THO components (Hpr1, Mft1, Thp2, Tho2, Tex1) (Chavez et al., 2000) that have a role in transcription elongation (Rondon et al., 2003), the mRNA export adaptors Sub2 and Yra1, and the SR-like proteins Gbp2 and Hrb1 (Hurt et al., 2004; Reed and Cheng, 2005; Strasser et al., 2002) (Figure 1A). The THO sub-complex and the mRNA export adaptor Yra1 interact with S2-S5 phosphorylated CTD (MacKellar and Greenleaf, 2011; Meinel et al., 2013) allowing their recruitment during transcription elongation.
TREX co-transcriptional loading by RNA PolII is proposed to facilitate subsequent interaction of Sub2, Yra1, Gbp2 and Hrb1 with the nascent transcripts.
mRNA 3’ end processing
The recruitment of the 3’end cleavage and polyadenylation factor (CPF) as well as the Cleavage factor 1A (CF1A) depends on the polyadenylation signal but also on a specific CTD phosphorylation pattern. Indeed, the CF1A components Pcf11 and Cft1, as well as the CPF 3’ end processing factors Rna14, Rna15, and Cft2 interact with S2
phosphorylated CTD, (Barilla et al., 2001; Dichtl et al., 2002; Kyburz et al., 2003;
Noble et al., 2005). 3’ end cleavage and polyadenylation is directly linked to transcription termination as defects in 3’ end formation result in transcriptional readthrough (Birse et al., 1998); 3’end formation is also tightly coupled to mRNA export, as defects in cleavage and polyadenylation result in mRNA export block (Hilleren et al., 2001). Notably, Pcf11 recruits the mRNA export adaptor Yra1 during elongation; Yra1 binding to Pcf11 contributes to poly(A) site choice by preventing premature interaction of Pcf11 with the CF1A component Clp1 (Bentley, 2014;
Proudfoot et al., 2002). At a later stage of transcription, interaction of the ATP- dependent RNA helicase Sub2 with Yra1 disrupts the Pcf11-Yra1 interaction allowing the Pcf11-Clp1 interaction to occur; concomitantly, Yra1 is transferred to the transcript together with Sub2 (Figure 1B) (Johnson et al., 2009a; Johnson et al., 2011a). Finally, THO-Sub2 were shown to stimulate 3’ end formation and mRNP release from the transcription site (Rougemaille et al. 2008). Together all these observations indicate tight functional coupling between transcription, 3’ mRNA processing and assembly of a mature and export competent mRNP complex.
mRNP surveillance and export
Coupling between transcription and export is further highlighted by the recruitment of the general mRNA export receptor Mex67/Mtr2 complex during transcription elongation via the interaction of the UBA-domain of Mex67 with the ubiquitinated form of the THO component Hpr1 (Figure 1C) (Dieppois et al., 2006;
Gwizdek et al., 2006; Hobeika et al., 2007; Segref et al., 1997). At a later step, the association of Mex67 with mRNPs is mediated by mRNA export adaptor proteins such as Yra1, the SR like protein Npl3, and the poly(A) binding protein Nab2 (Figure 1A) (Fasken et al., 2008; Gilbert et al., 2001; Iglesias et al., 2010; Reed and Hurt, 2002;
Segref et al., 1997; Stutz and Izaurralde, 2003). Nab2 binds poly(A) tails after transcript cleavage and poly(A) polymerase action (Schmid et al., 2015).
mRNP assembly in the nucleus is subjected to tight quality control by the nuclear exosome, which subunit Rrp6 triggers the 3’-5’ exonucleolytic degradation of malformed mRNP complexes prior to mRNA export. (Figure 1A) (Hilleren et al., 2001). Upon Nab2 depletion, for instance, poly(A) transcripts are degraded by the nuclear exosome leading to a bulk mRNA decay (Schmid et al., 2015). The quality control process by the nuclear exosome is also a co-transcriptional event. Degradation of malformed transcripts by Rrp6 is stimulated by the non-canonical polyadenylation TRAMP complex which adenylates malformed mRNPs and acts as a co-factor in mRNA surveillance (Callahan and Butler, 2010; Rougemaille et al., 2007).
A final mRNP quality control step occurs at the nucleoplasmic face of the pore and involves the NPC-associated Mlp1 and Mlp2 Myosin-like filamentous proteins.
Mlp1 was shown to retain faulty intron-containing pre-mRNAs (Galy et al., 2004;
Vinciguerra et al., 2005). Mlp1/2 also interact with the poly(A) binding protein Nab2 establishing another link between mRNP surveillance and the nuclear periphery (Fasken et al., 2008; Tutucci and Stutz, 2011). Furthermore, studies from our laboratory indicated that ubiquitination of Yra1 by the E3 ligase Tom1 displaces Yra1 from mRNPs, an event that is part of an mRNP surveillance step functionally linked to Mlp2 prior to export (Iglesias et al., 2010) (Figure 2A).
Properly assembled export competent mRNPs are translocated through the central channel of the nuclear pore complex via interaction of the export receptor Mex67 with a specific class of NPC proteins containing repeats rich in phenylalanines (F) and glycines (G) called FG-Nups (Terry and Wente, 2007) (Figure 2B). The
directionality of mRNP through the NPC is ensured by Dbp5, a nucleo-cytoplasmic shuttling ATPase that becomes associated with mRNPs in the nucleus. On the cytoplasmic face of the NPC, interaction of Dbp5 with the NPC protein Gle1 bound to its co-factor IP6 triggers Dbp5 ATPase activity, resulting in mRNP conformational changes and release into the cytoplasm. The export receptor Mex67/Mtr2 and mRNA binding protein Nab2 dissociate from the mRNP and are recycled back into the nucleus (Figure 2B) (Ledoux and Guthrie, 2011; Lund and Guthrie, 2005; Tran et al., 2007).
Figure 1: Coupling of transcription, processing and mRNA export.
The main steps of transcription are shown. A) Shortly after initiation, the transcript is capped, a process facilitated by RNA PolII CTD phosphorylation on Ser5 (Cho et al., 1997; McCracken et al., 1997a).
During transcription elongation, the THO complex is recruited to the transcribing RNA PolII through interaction with Ser2-Ser5 phosphorylated CTD (Rondon et al., 2003); Yra1 is recruited via interaction with the 3’end processing factor Pcf11 bound to the phosphorylated CTD (MacKellar and Greenleaf, 2011). During elongation, the Sub2 RNA helicase interacts with THO and promotes the recruitment of Yra1 to form the TREX complex. Displacement of Yra1 allows Pcf11 to recruit the 3’end processing complex CF1A through interaction with Clp1 (Johnson et al., 2011a) and detailed in (B). During elongation, the mRNA export receptor Mex67/Mtr2 is recruited via interaction with ubiquitylated Hpr1 (C). At the termination/3’end processing step, Sub2 induces a conformational change promoting dissociation of the cleavage and polyadenylation complex, an event paralleled by the binding of Yra1 (and other mRNA export adaptors such as Nab2 and Npl3) as well as the Mex67/Mtr2 receptor on the mRNA (Dieppois et al., 2006; Gwizdek et al., 2006; Hobeika et al., 2007; Segref et al., 1997). Export competent mRNPs are released from the transcription site under the quality control of the exosome (Hilleren et al., 2001). The splicing events occurring during transcription elongation are not shown.
Figure modified from (Oeffinger and Zenklusen, 2012).
Figure 2: mRNP recruitment at the Nuclear Pore.
A) mRNP complexes associate with nuclear pores through interaction of mRNA export adaptor proteins and export receptor Mex67/Mtr2 with Mlp1/2 and other NPC basket proteins. In this context, ubiquitination by Tom1 promotes Yra1 dissociation from mRNPs acting as a license for export (Iglesias et al., 2010). Inducible genes relocate to the nuclear periphery through interaction of the SAGA co- activator complex with the NPC-bound TREX2 complex (gene gating model) (Brickner and Walter, 2004; Cabal et al., 2006; Taddei et al., 2006).
B) mRNP translocation through the nuclear pore depends on the interaction of Mex67/Mtr2 with FG- Nups (in green) (Terry and Wente, 2007). The release of Mex67/Mtr2 and Nab2 from the mRNP on the cytoplasmic site is triggered by the ATPase activity of Dbp5 stimulated by Gle1-IP6 (Ledoux and Guthrie, 2011; Lund and Guthrie, 2005; Tran et al., 2007). Figure modified from (Oeffinger and Zenklusen, 2012).
Gene gating and gene looping at Nuclear Pores
A certain number of highly expressed or highly induced genes, especially those dependent on the SAGA co-activator complex, relocate to the nuclear periphery upon activation in a process dependent on SAGA components as well as TREX2, an evolutionary conserved complex associated with the nuclear side of the NPC and consisting of Sac3, Thp1, Cdc31 and Sus1 (Figure 2A) (Brickner and Walter, 2004;
Cabal et al., 2006; Taddei et al., 2006). Sus1 was proposed to play an important role in this “gene gating” process (Blobel, 1985) as its loss interferes with NPC gene anchoring (Cabal et al., 2006). Besides Sac3, Sus1 also interacts with the SAGA co- activator subunit Ubp8, and binds Ser2 and Ser5 phosphorylated CTD (Jani et al., 2009;
Pascual-Garcia et al., 2008; Rodriguez-Navarro et al., 2004). Thus, Sus1 may act as a bridge connecting transcribing genes to nuclear pores. More recently, TREX2 was also
proposed to influence gene positioning and activity through interaction with the Mediator complex, an essential regulator of RNA PolII, further increasing the interaction network between active genes and the nuclear periphery (Schneider et al., 2015).
The mRNA export receptor Mex67 has also been implicated in NPC gene anchoring as it is co-transcriptionally recruited to active genes through interaction with the THO elongation complex (Dieppois et al., 2006; Gwizdek et al., 2006) and together with Mtr2 interacts with Sac3 (Fischer et al., 2002), that is part of the TREX2 complex tethered to the NPC through binding to the nucleoporin Nup1 (Jani et al., 2014).
Besides Nup1, other nucleoporins located at the NPC nuclear basket have been involved in the interaction with mRNPs or activated genes, based on the loss of peripheral localization in specific mutant backgrounds (Cabal et al., 2006; Dieppois et al., 2006; Texari et al., 2013). Nup2 was found to be associated at promoters of transcribing genes (Schmid et al., 2006); Nup60 indirectly influences gene anchoring as it participates in NPC association of Mlp1 and Mlp2, implicated in the binding of the TREX2 component Sac3 (Lei et al., 2003) and of SAGA constituents (Luthra et al., 2007) (Figure 3).
Chromosome conformation capture (3C) experiments have shown that gene loops can form through interaction of 5’ and 3’ gene regions and that these loops may be influenced by NPC components such as Nup2 or Mlp proteins (Casolari et al., 2005;
Schmid et al., 2006; Tan-Wong et al., 2009). Is not clear whether gene looping occurs before gene gating, or whether NPC association stabilizes the loops. Gene looping depends on the interaction between mRNA transcription and processing factors located at the 5’ and 3’ ends of activated genes (Ansari and Hampsey, 2005; O'Sullivan et al., 2004; Singh and Hampsey, 2007). It has been proposed that this gene conformation improves gene expression by favoring the recycling of RNA PolII from the 3’ to 5’ end of genes. However, no strong effect on gene expression has been reported when gene loops were abrogated (Ansari and Hampsey, 2005). One reported outcome of gene looping is the transcriptional memory effect, which allows faster reactivation of inducible genes after short-term repression (Brickner et al., 2007; Brickner et al., 2010;
Laine et al., 2009; Tan-Wong et al., 2009). More recently, gene loop formation was proposed to influence transcription directionality of bidirectional promoters by favoring the transcription of the coding region (Castelnuovo and Stutz, 2013;
Grzechnik et al., 2014; Tan-Wong et al., 2012).
The Nuclear Pore Complex
The Nuclear Pore Complex (NPC) allows molecular exchanges between the nuclear and cytoplasmic compartments in a selective and bidirectional process (Strambio-De-Castillia et al., 2010). The NPC is a large 60-125 MDa proteinaceous cylindrical complex arranged around a central channel inserted into the nuclear envelope. The NPC presents an octagonal symmetry around the central axis and its overall structure is conserved from yeast to higher eukaryotes (DeGrasse et al., 2009) (Figure 3). A variety of biochemical and microscopy approaches, including immuno- electron microscopy, cryo-electron tomography and crystallography allowed to divide the NPC into a central spoke ring assembly framed by a cytoplasmic ring from which emanate eight cytoplasmic filaments and a nuclear ring from which protrude eight filaments that converge into a distal ring forming the nuclear basket (Alber et al., 2007;
Rout et al., 2000) (Figure 3).
The NPC is constituted of 30 different nucleoporins or Nups, organized in a number of sub-complexes each present in 8-16 copies to reach a total of 400-500 proteins per NPC (D'Angelo and Hetzer, 2008). While a subset of Nups is located on both sides of the NPC, others present an asymmetric distribution and are preferentially associated with the cytoplasmic or nuclear side of the NPC. Among the 30 different Nups, 15-20 belong to the class of FG-nucleoporins characterized by unstructured domains rich in Phe-Gly repeats that act as docking sites for transport receptors, such as Mex67/Mtr2, bound to their cargoes. A fraction of FG-Nups is part of the central ring, with the FG-repeat domains protruding into the central channel, while other FG- Nups present an asymmetric distribution and are associated either with the cytoplasmic filaments or the nuclear basket. Nucleo-cytoplasmic transport depends on the sequential interaction of import or export complexes with FG-repeat containing Nups spanning the pore (Alber et al., 2007; Bayliss et al., 2000; Cronshaw et al., 2002; Radu et al., 1995; Rout et al., 2000; Terry and Wente, 2007).
More specifically, the asymmetric FG-Nups Nup42 and Nup159 that are part of the cytoplasmic filaments have been implicated in mRNA export. Indeed, these filaments contain binding sites for Gle1 and the RNA helicase Dbp5, two factors pivotal for mRNA release into the cytoplasm and for the first steps of mRNA
translation (Ledoux and Guthrie, 2011; Stelter et al., 2007) and see above. Furthermore Nup159 interacts also with the Dynein component Dyn2, binding that contributes to the cytoplasmic filamentous structure (Stelter et al., 2007). Nup60 and Nup1 are asymmetric FG-Nups associated with the nuclear basket (Strambio-De-Castillia et al., 2010), which contribute to the association of the filamentous part of the nuclear basket mainly composed of the myosin-like proteins Mlp1 and Mlp2 (Tpr in higher eucaryotes) (Strambio-de-Castillia et al., 1999) (Figure 3). Mlp2 arose from a gene duplication event and is also found in association with Spindle Pole Bodies (Niepel et al., 2005). The Nup2 FG-Nup only transiently associates with the nuclear basket and behaves more like a transport factor (Dilworth et al., 2001) (Figure 3). The importance of nucleoporins in mRNA export and mRNP quality control was discussed in previous sections.
Figure 3: Conserved structure of the nuclear pore complex.
The nuclear pore complex is a cylindrical structure presenting an eight-fold symmetry around the central axis and consisting of the nuclear face, the central channel, and the cytoplasmic face. The boxes contain the list of yeast nucleoporins present in each part with the corresponding vertebrate homologues. The Nups discussed in the text are highlighted. Figure from (Strambio-De-Castillia et al., 2010).
Nuclear Organization
Eukaryotic genomes are organized non-randomly inside the nucleus creating functional subnuclear compartments. The importance of the distribution of DNA loci relative to the nuclear periphery in transcription repression and activation has been extensively investigated. In the last decade, thanks also to improved microscopy techniques, nuclear organization has not only been implicated in transcription, but also in DNA replication and repair events. Overall the faithful interplay between these processes in relation with the nuclear periphery contributes to maintain genome stability (Bermejo et al., 2012; Chan et al., 2014; Mekhail and Moazed, 2010; Nagai et al., 2011).
The nucleus of S. cerevisiae is smaller (~1µm diameter) in size than the nucleus of mammalian cells (~10µm diameter). Despite its small size, extensive analysis using microscopy techniques and 3D reconstitution imaging allowed to reveal the main characteristics of chromatin organization in yeast nuclei. Unique features of budding yeast nuclear organization, absent in higher eukaryotes, comprise a closed mitosis with no nuclear envelope breakdown, the lack of a nuclear lamina network associated with the inner nuclear membrane, as well as spindle pole bodies (SPBs) that remain embedded within the nuclear envelope during all the cell cycle (Webster et al., 2009).
The chromatin inside the nucleus is subjected to different levels of compaction with heterochromatin corresponding to condensed transcriptionally inactive domains, and euchromatin corresponding to decondensed actively transcribed regions. Furthermore, the chromatin is not only differently packed inside the nucleus but also organized in distinct territories. Indeed budding yeast chromosomes adopt the so-called Rabl configuration, in which all centromeres cluster at the SPB throughout the cell cycle;
FISH and immunofluorescence microscopy showed that chromosome arms extend away from the centromeres/SPB and that the heterochromatic telomeric and subtelomeric regions colocalize in several discrete foci associated with the nuclear envelope in regions mostly distinct from nuclear pores (Figure 4) (Bystricky et al., 2005; Gotta et al., 1996; Taddei and Gasser, 2012). Accordingly, chromosome conformation capture (3C) experiments combined with massive sequencing reveal centromere clustering around Spindle Pole Bodies and the occurrence of interactions between telomeres of the same or different chromosomes provided that the chromosome arms are of similar length, consistent with the Rabl-like configuration
(Duan et al., 2010; Hediger and Gasser, 2002; Rodley et al., 2009; Therizols et al., 2010).
Figure 4: Rabl-like configuration of yeast chromosomes.
The Rabl-like configuration of yeast chromosomes is established during Telophase and persists in Interphase. Clustered centromeres (red dots) interact with SPBs thanks to microtubule anchorage. The heterochromatic subtelomeric and telomeric chromosomal regions (green dots) are anchored to the nuclear envelope. The nucleolus containing the rDNA locus (shown in yellow) is located opposite the CEN/SPB. Figure from (Taddei and Gasser, 2012).
The yeast ribosomal DNA array on Chromosome XII is contained within the nucleolus, a crescent shaped compartment occupying one third of the nucleus and located next to the nuclear envelope opposite to the SPB (Leger-Silvestre et al., 1999;
Yang et al., 1989). The nucleolus is the site of ribosome biogenesis through RNA PolI and PolIII transcription of the 9.1 kb rDNA sequence. The 150-200 rDNA tandem repeats are prone to homologous recombination events that could be deleterious for rDNA stability and cause premature senescence (Sinclair et al., 1997). Two strategies have been acquired to limit recombination in the nucleolus. The first depends on the deacetylase Sir2 that promotes nucleosome compaction and silencing (Smith and Boeke, 1997); the second depends on the CLIP complex that keeps the nucleolus anchored to the nuclear envelope (Mekhail et al., 2008).
The nuclear envelope proteins Esc1 and Mps3 provide additional docking sites, distinct from nuclear pore complexes, that are crucial for chromatin organization (Figure 5). Esc1 contributes to the association of repressed telomeric chromatin to the nuclear envelope through interaction with the Sir4 silencing protein bound to the telomeric binding protein Rap1 (Andrulis et al., 2002; Taddei et al., 2004). The interaction of Sir4 with the nuclear membrane SUN-protein Mps3 represents an
alternate pathway for tethering silent subtelomeric regions to the nuclear periphery (Bupp et al., 2007).
There is another silencing-independent mechanism that anchors telomeric regions at the nuclear envelope, preferentially during S-phase. In this case, Mps3 interacts with the telomerase subunit Est1 bound to telomeres via the yKu70/Ku80 heterodimer (Schober et al., 2009). Some of these interactions are cell cycle regulated and depend on protein SUMOylation (Ebrahimi and Donaldson, 2008; Ferreira et al., 2011). Notably, the yKu80-Mps3 interaction depends on yKu80 SUMOylation by the E3 ligase Siz2 during S-phase (Ferreira et al., 2011); in G1, the tethering of yKu proteins is independent of Mps3 and involves a yet unidentified anchor (Schober et al., 2009; Taddei and Gasser, 2012; Taddei et al., 2004).
Interestingly some telomeres also associate with nuclear pores and this localization is essential to repair double strand breaks (DSBs) in the corresponding subtelomeric regions (Therizols et al., 2006).
Figure 5: Yeast nuclear territories and nuclear periphery domains.
Shown are the nuclear domains with active or repressive states of RNA PolII transcription. The NPC (blue) is associated with actively transcribed chromatin. The repressed subtelomeric and telomeric regions (green) tend to associate with the nuclear envelope. The nucleolus is shown in yellow. The SPB associated with centromeres is shown in red. The box on the right illustrates the thethering of telomeric regions to the nuclear envelope through interaction with the Esc1 and Mps3 docking sites. See text for details. Figure modified from (Zimmer and Fabre, 2011).
Nuclear Dynamics during Genome Instability
Eroded telomeres tethered at the nuclear envelope
The tethering at the nuclear envelope of highly repeated genomic loci, such as the 9.1 Kb rDNA tandem repeats and the 300 bp arrays of telomeric TG1-3 repeats, plays an important role in preventing aberrant recombination events that could be detrimental to genome stability. Accordingly, hyper-recombination phenotypes were observed at eroded telomeres formed in absence of the ATM kinase Tel1, since they are not anymore anchored at the Nuclear Envelope by Mps3 (Schober et al., 2009).
Eroded telomeres in telomerase negative cells are localized at Nuclear Pores instead of being anchored at the nuclear envelope (Khadaroo et al., 2009). The relocation to nuclear pores depends on the SUMOylation by Siz1/Siz2 of the replication protein A (RPA), which is bound to resected telomeres and mediates the interaction of eroded telomeres with the SUMO targeted Ubiquitin ligase (STUbL) Slx5-Slx8 (Churikov et al., 2016), located at the NPC through association with Nup84 (Nagai et al., 2008). The interaction of eroded telomeres with Slx5-Slx8 at the nuclear pore triggers type II telomere recombination, during which TG1-3 repeats are amplified in a process dependent on Rad52 and Sgs1 (Churikov et al., 2016). Because Slx5-Slx8 interact with a subunit of the 26S proteasomal lid (Krogan et al., 2006), it has been speculated that the Slx5-Slx8-dependent nuclear localization of SUMOylated telomere bound proteins leads to their Ubiquitin-dependent proteasomal degradation. Indeed, Slx5-Slx8 are evolutionary conserved RING finger proteins that can recognize SUMOylated targets through multiple SUMO interaction motifs (SIM) and subsequently trigger Ubiquitination through their RING domains important for dimerization and ubiquitin ligase activity (Hickey et al., 2012; Sriramachandran and Dohmen, 2014). Furthermore it was proposed that SUMOylated telomere bound proteins targeted to the NPC could be de-SUMOylated by the SUMO protease Ulp1 located at the nuclear basket (Zhao et al., 2004), since efficient type II telomere recombination requires Ulp1 localization at the Nuclear Pore (Churikov et al., 2016) (Figure 6 ).
Figure 6: Eroded telomeres are targeted to nuclear pores.
Proteins bound to eroded telomeres are SUMOylated by Siz1/Siz2 and recruited to the Nuclear Pore via the STUbL Slx5/Slx8. SUMOylated proteins are removed possibly through proteasomal degradation (not shown) or through de-SUMOylation by the SUMO protease Ulp1 located at the Nuclear Pore (not shown). These events trigger repair and telomere repeat amplification by type II recombination. Image modified from (Horigome and Gasser, 2016).
Persistent DNA double strand break relocation at the nuclear envelope Similar chromatin dynamics mechanisms have been described for persistent Double Strand Breaks (DSBs) that cannot be repaired by homologous recombination (HR) (Nagai et al., 2008) (Figure 7). It was shown that depending on the cell cycle phase, irreparable DSBs are either recruited to nuclear pores or to the nuclear envelope through interaction with Mps3 (Horigome et al., 2014). In G1 and S phase, proteins bound to irreparable DSBs are poly-SUMOylated by the E3 ligases Siz2 and Mms21 promoting interaction with the STUbL Slx5-Slx8 at nuclear pores (Horigome et al., 2016). In S phase, proteins bound to irreparable DSBs and mono-SUMOylated by Mms21 relocate the persistent DSB to the Mps3 docking site independently of Slx5- Slx8 (Horigome et al., 2016). The authors speculate that the irreparable DSB relocation at the nuclear pore leads to the proteasomal degradation of proteins targeted by the STUbL Slx5-Slx8 (Nagai et al., 2008), an event important to induce alternative repair pathways such us Break Induced Replication (BIR) (Horigome et al., 2016). The clustering or sequestration of irreparable DSBs at Mps3 (Kalocsay et al., 2009; Oza et al., 2009) induced by mono-SUMOylation during S-phase (Horigome et al., 2016;
Horigome et al., 2014) is explained as a cell strategy to prevent ectopic Homologous Recombination.
Figure 7: The recruitment of irreparable DSBs to Nuclear Pores or Mps3 docking sites depends on SUMOylation.
Persistent DSBs are recruited to the nuclear pore in G1 and S phase through poly-SUMOylation of DSB- bound proteins recognized by Slx5-Slx8. This could lead to proteasomal degradation and non-canonical recombination by Break Induced Replication (BIR). In S phase, mono-SUMOylation of DSB-bound proteins results in the recruitment to Mps3 docking sites at the nuclear envelope likely to prevent aberrant Homologous Recombination. Figure from (Horigome and Gasser, 2016).
In contrast to persistent DSBs, repairable DSBs can be repaired by Non Homologous End Joining (NHEJ) when they occur in G1, and Homologous Recombination (HR) when they occur in S and G2 phase. NHEJ is an error prone
repair pathway since it ligates the two DNA extremities without using a homologous template, while HR is an error free pathway since it uses the homologous DNA strand for the repair (Figure 8).
Figure 8: DSB repair by NHEJ in G1 phase or HR in S and G2 phase.
In yeast, Double Strand Breaks are recognized by the MRX complex (Mre11-Rad50-Xrs2). In G1, the Non Homologous end Joining (NHEJ) pathway repairs DSBs thanks to the recruitment of the Ku70- Ku80 complex and the ligation by Dnl4-Lif1-Nei1. In S and G2 phase, DSBs are repaired by Homologous Recombination (HR). The MRX complex and Sae2 enzyme start the 5’!3’ exonucleolytic process. Extensive resection is mediated by the exodeoxyribonuclease 1 (Exo1) and the Dna2-Sgs1 DNA-end processing enzyme. The generated DNA single strands are coated by the Recombination Protein A (RPA) complex; subsequently Rad52 displaces RPA with Rad51 allowing initiation of strand invasion and Homologous Recombination to occur. Figure modified from (Papamichos-Chronakis and Peterson, 2013).
Efficient repair by HR correlates with increased mobility of DSBs in the nucleus especially in diploid cells, in which overall chromatin mobility is enhanced
presumably to favor the search for homologous sequences (Mine-Hattab and Rothstein, 2012).
In haploid cells a single DSB or treatment with low levels of zeocin (DSB- causing drug) do not change genome-wide chromatin dynamics although the damaged locus shows increased intranuclear mobility (Dion et al., 2012). In contrast, prolonged exposure to Zeocin (up to 6h), which causes damage in 60% of the cells, increases overall chromatin dynamics (Herbert et al., 2017). Furthermore, DSBs generated by collapsed replication forks do not show increased chromatin movement, reflecting the absence of need to search for homologous sequences to be repaired, since the template is already present on the sister chromatid (Dion et al., 2012). These observations indicate that chromatin mobility depends on the type of DNA damage and favored repair process.
Finally, it was observed that multiple DSBs cluster together in repair centers characterized by the co-localization with the repair factor Rad52, a situation likely favored by increased chromatin movements (Lisby et al., 2003) (Figure 9).
Interestingly, spontaneous S-phase damage recognized by Rad52 shows reduced mobility and is located within the nucleus, consistent with the mobility of DSBs repaired based on sister chromatid homology (Dion et al., 2013). Notably, when a DSB occurs within the rDNA locus, it has to move outside the nucleolus to form a Rad52 focus and be repaired. If repair occurred within the nucleolus by HR, it would lead to rDNA hyperrecombination and excision of rDNA circles (Torres-Rosell et al., 2007).
Figure 9: Changes in Chromatin dynamics upon DNA damage and formation of Rad52 foci, repair centers for HR.
Upon DNA damage, DSBs cluster in repair centers characterized by co-localization of Rad52, a repair protein important for HR to occur. Rad52 foci always form in the nucleoplasm or nuclear interior, next to the nucleolus, also when the DSB is formed at the rDNA locus (nucleolus in yellow). Image modified from (Lebeaupin, 2015)
The mRNA export adaptor Yra1
Yra1 (Yeast RNA annealing protein 1) is an essential protein in S.
cerevisiae, well characterized as an mRNA export adaptor involved in transcription elongation, 3’ processing, and finally mRNA export together with the Mex67/Mtr2 export receptor and the poly(A) binding protein Nab2 (as described above). Yra1 was initially purified as a protein with strong RNA annealing activity using an in vitro assay (Portman et al., 1997), however it is still unclear whether RNA annealing activity is relevant for Yra1 function in vivo.
Yra1 is an evolutionarily conserved protein from yeast to human (Preker and Guthrie, 2006; Strasser et al., 2002) and belongs to the RNA and Export Factor (REF) family of hnRNP-‐like proteins (Portman et al., 1997; Stutz et al., 2000). REF proteins present a conserved domain organization with a central RNP-‐motif containing RNA binding domain (RBD) and two highly conserved N-‐ and C-‐
terminal boxes (N-‐box and C-‐box). These highly conserved domains are separated by two more variable regions (N-‐var and C-‐var), rich in glycines, serines and positively charged amino acids that are related to RGG boxes found in many other RNA binding proteins (Stutz et al., 2000); (Burd and Dreyfuss, 1994) (Figure 10).
RNA binding is mediated by the N-‐var and C-‐var domains and not the RBD, which is not essential for growth. Moreover, yra1 mutants lacking the whole N-‐terminal or C-‐terminal (N-‐box+N-‐var or C-‐box+C-‐var) regions are also viable indicating some functional overlap. At least one highly conserved N-‐box or C-‐box is required for viability as deletion of both is lethal (Zenklusen et al., 2001).
Interestingly, the same Yra1 domains can interact with different partners (Figure 10), possibly regulating the timing of Yra1 function in mRNP processing through mutually exclusive interactions.
Figure 10: Yra1 domains and partners.
Yra1 protein domains characteristic of the REF protein family with corresponding interacting factors are shown based on (Iglesias et al., 2010; Johnson et al., 2009a; Ma et al., 2016; Strasser and Hurt, 2001; Stutz et al., 2000; Zenklusen et al., 2001).
Among the partners of Yra1, both the mRNA export receptor Mex67 and the DEAD-‐box RNA helicase Sub2 interact with the N-‐terminal (N box + N-‐var) and C-‐terminal (C-‐box + C-‐var) regions of Yra1, indicating a certain level of redundancy between these domains (Strasser and Hurt, 2001; Zenklusen et al., 2001). The N-‐terminal region also interacts with Pcf11, a component of Cleavage Factor 1A (CF1A) involved in mRNA 3’ end processing. Notably, inactivation of Pcf11 leads to decreased recruitment of Yra1 on transcribed loci without affecting the binding of Sub2 (Johnson et al., 2009a). Together these observations led to a model in which Yra1 is recruited during transcription elongation through interaction with Pcf11, while Sub2 is recruited independently through interaction with the THO component Hpr1. In a subsequent step, interaction with Sub2 promotes the loading of Yra1 on the nascent transcript/elongation complex forming the TREX complex. The export receptor Mex67 is also recruited co-‐
transcriptionally through interaction with the ubiquitinated Hpr1 THO component (Dieppois et al., 2006; Gwizdek et al., 2006). At the end of the gene, concomitant with 3’ end processing, Sub2 induces a rearrangement resulting in the interaction of Yra1 with Mex67 on the processed mRNP, allowing its release and export towards the cytoplasm (Johnson et al., 2009a; Johnson et al., 2011a;
Tutucci and Stutz, 2011; Zenklusen et al., 2002) (Figure 11). Interestingly recent
