Characterization of the RNA in Insect Cells

Role in mRNA Surveillance

Viktoria Hessle

Doctoral Thesis Stockholm, 2011 Department of and Functional Genomics Stockholm University, Sweden

The cover picture shows an immunolabeling experiment with an antibody against the core exosome Rrp4 in a salivary gland of Chironomus tentans.

 Viktoria Hessle, 2011 ISBN 978-91-7447-208-0 Printed in Sweden by Universitetsservice US-AB Stockholm 2011 Distributor: Stockholm University Library

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To my family

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Table of Contents ABSTRACT ...... 4 LIST OF ARTICLES INCLUDED IN THIS THESIS ...... 5 LIST OF ABBREVIATIONS...... 6 INTRODUCTION...... 7 NUCLEAR ORGANIZATION IN EUKARYOTIC CELLS...... 7 The nuclear envelope, chromatin and the ...... 7 Subnuclear compartments ...... 10 EXPRESSION OF PROTEIN-CODING IN EUKARYOTIC CELLS ...... 15 Co-transcriptional mRNA processing ...... 16 Coupling between the processing events ...... 20 mRNA surveillance ...... 21 THE EXOSOME ...... 23 Exosome complexes in and archea...... 23 The exosome in ...... 25 The catalytic subunits of the exosome ...... 26 The exosome in the nucleus ...... 27 The exosome in the ...... 29 Activation of the exosome ...... 30 The role of the nuclear exosome in mRNA surveillance...... 31 PRESENT INVESTIGATION...... 33 Aim of this thesis...... 33 Model systems...... 34 SUMMARY OF THE PROJECTS INCLUDED IN THIS THESIS...... 36 CONCLUSIONS...... 41 ACKNOWLEDGEMENTS...... 43 REFERENCES ...... 44

3 Abstract

Eukaryotes have evolved quality control systems to prevent the synthesis of defective that are potentially damaging for the cell. The exosome is an evolutionarily conserved multiprotein complex that is found in both archea and eukaryotes. The exosome has exoribonucleolytic activity and is one of the key players in quality control. However, little is known about the molecular mechanisms by which the exosome functions in metazoans. The main objective of this thesis has been to study the exosome and its role in nuclear mRNA surveillance using the diptera Chironomus tentans (C. tentans) and Drosophila melanogaster (D. melanogaster) as model systems. To characterize the exosome in C. tentans, we have cloned partial cDNAs encoding the C. tentans orthologs of Rrp4 and Rrp6, we have raised antibodies against these two exosome subunits, and we have used a combination of techniques including , immuno-electron microscopy, chromatin , immunoprecipitation and . We have studied the core exosome subunit Rrp4 and found that it is associated with transcribed genes in the polytene chromosomes of C. tentans. We have also found that a significant fraction of Rrp4 is associated to nascent pre-mRNPs. We have been interested in understanding the mechanism by which the exosome is recruited to transcriptionally active genes, and we show that several exosome subunits including Rrp4 interact in vivo with the mRNA-binding protein Hrp59/hnRNP M. Depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for a stable association of the exosome with nascent pre-mRNPs. Our results on Rrp4 support a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized through direct interactions with specific hnRNP proteins. We have shown that the catalytic exosome subunit Rrp6 is also recruited to transcriptionally active genes and interacts with nascent pre-mRNPs. Moreover, we show that Rrp6 interacts with mRNPs in transit from the to the nuclear pore complex, and is released from the mRNP at the pore during early stages of nucleo-cytoplasmic translocation. These observations suggest that Rrp6 is not only part of the co-transcriptional surveillance but also surveys the mRNP during nucleoplasmic transport. Immunofluorescent stainings have revealed that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies (RNBs) colocalize with SUMO whereas in D. melanogaster we have identified RNBs and SUMO-rich bodies as different entities. The RNBs in C. tentans do not contain Rrp4 and are therefore related to exosome-independent activities of Rrp6. In another series of experiments, we have developed an in vivo system to study the co- transcriptional surveillance of mRNA biogenesis. We have constructed D. melanogaster S2 cells expressing human β-globin genes, either wild type of with mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that at least two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that several mechanisms contribute to co- transcriptional surveillance in insects, being RNA degradation by the exosome perhaps not the major one.

4 List of articles included in this thesis

This thesis is based on the following articles, which will be referred to with their Roman numerals.

Paper I:

Bothelo SC, Tyagi A, Hessle V, Farrants AK, Visa N. (2008). The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation. Insect Mol Biol. Sep;17 (5): 505-13.

Paper II:

Hessle V, Björk P, Sokolowski M, González de Valdivia E, Silverstein R, Artemenko K, Tyagi A, Maddalo G, Ilag L, Helbig R, Zubarev RA, Visa N. (2009) The exosome associates cotranscriptionally with the nascent pre-mRNP through interactions with heterogeneous nuclear ribonucleoproteins. Mol Biol Cell. Aug;20 15):3459-70

Paper III:

Eberle A, Hessle V, Helbig R, Dantoft W, Gimber N, Visa N. (2010). Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing- defective genes. PLoS One. Jul 12;5 (7):e11540.

Paper IV (manuscript):

Hessle V, von Euler A, González de Valdivia E, Visa N. The intranuclear localization of the Rrp6 in insect cells.

The articles were reproduced with permission from the copyright holders.

5 List of abbreviations

BR Balbiani Ring BR-mRNP Balbiani Ring messenger Ribonucleoprotein ChIP Chromatin Immuno-Precipitation CPSF Cleavage and Specificity Factor CstF Cleavage stimulatory Factor CTD C-Terminal Domain (of RNA polymerase II) CUT Cryptic Unstable Transcript Da Dalton EM Electron Microscopy EJC Exon-Junction Complex FISH Fluorescent In Situ Hybridization GFP Green Fluorescent Protein hnRNP heterogeneous nuclear Ribonucleoprotein IEM Immuno Electron Microscopy IF Immuno-Fluorescence IP Immuno-Precipitation kb kilobasepair mAb monoclonal Antibody mRNA messenger Ribonucleic Acid mRNP messenger Ribonucleoprotein NB Nuclear Body NMD Nonsense-Mediated Decay NPC Nuclear Pore Complex N-terminal amino terminal ORF Open Reading Frame QPCR Quantitative Polymerase Chain Reaction PAP Poly (A) Polymerase Pre-mRNA Pre-messenger Ribonucleic Acid RNase RNB Rrp6-rich Nuclear Bodies RNP Ribonucleoprotein TRAMP Trf4-Air1/2-Mtr4p Polyadenylation Complex TREX Transcription and Export Complex

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Introduction

This thesis aim to scrutinize the RNA exosome, how the exosome is involved in co-transcriptional messenger RNA (mRNA) surveillance or quality control in eukaryotic cells, primarily in the cells of Chironomus tentans but also in Drosophila melanogaster. Additionally, this thesis touches upon the field of nuclear organization of the eukaryotic cell. The primary goal for basic research is to learn about the fundaments of the human world. The questions approached by basic research often arise out of curiosity without commercial benefits. However, in the long run basic research often generates ideas and knowledge that can be used for applied purposes. A large part of the research done in the field of RNA exosome and nuclear organization has generated information about diseases such as . With the work included in this thesis I hope we have contributed to the mapping of the functions of the eukaryotic genome.

Nuclear organization in eukaryotic cells

Techniques such as light- and electron microscopic imaging have enabled the possibility to observe the functional cellular organization in situ. This has enriched our understanding of the highly dynamic nucleus of the eukaryotic cell. By chemical fixation of the nucleus it is possible to visualize proteins, to distinguish nuclear structures and to characterize distinct sub compartments. The introduction of live cell imaging has added the knowledge of the dynamics of proteins and chromatin. I will initially give a brief description of the basic constituents of the and of some of the nuclear sub compartments.

The nuclear envelope, chromatin and the nucleolus

Nuclear envelope The cell nucleus is a membrane-enclosed that harbors the genetic material of the cell. The nuclear envelope consists of a double lipid bilayer. The inner membrane is structurally supported by the nuclear lamina. This is a two-dimensional filamentous network built up by intermediate filaments made of proteins called lamins as well as other membrane-associated proteins. Besides providing support for the inner membrane, the nuclear lamina is involved in chromatin organization. The nuclear membrane is interrupted by a large number of pores, through which transport between the cytoplasmic- and nuclear compartments occurs. The pores are covered by large proteinacious structures forming the nuclear

7 pore complexes (NPCs). The NPC contains an aqueous channel that mediates all transport of molecules. The channel allows for passive transport of small molecules and signal-dependent and transport receptor-mediated active transport of larger molecules. The NPC is a conserved structure in eukaryotes. The main parts are the nuclear basket, the central core and the cytoplasmic fibrils. The nuclear part of NPCs is anchored to the nuclear lamina. The proteins in the NPC are generally referred to as nucleoporins or NUPs. Transport receptors interact with the phenylalanine and glycine repeats (FG- repeats) that characterize many of the NUPs. Yeast NPC consists of about 30 individual NUPs and several are present in multiple copies (reviewed by Strambio-De-Castillia et al., 2010).

Figure 1. Graphic overview of the eukaryotic cell nucleus describing the fundamental nuclear organization with chromosome territories, nuclear envelope and subnuclear compartments.

Chromatin The chromatin in the eukaryotic cell is composed of nucleosomes that are built up by four different histone proteins (H2A, H2B, H3 and H4) arranged in an octamer. An approximately 146 bp long stretch of DNA is wound twice around

8 the octamer. The nucleosomes are separated by “linker DNA”. The nucleosomes and the liker DNA stretches together constitute the typical “beads- on-a-string” appearance of the chromatin fiber. The length of the linker DNA varies, typically the distance is 20-60 bp. Local features such as specific DNA binding proteins and the flexibility of the DNA helix at certain DNA sequences affect the positioning of nucleosomes. The “beads- on-a-string” or 11-nm fiber is usually further folded into a 30-nm fiber, where the nucleosomes are packed upon one another with the help of histone H1 that interconnects the nucleosomes (reviewed by Widom 1989). The interphase nucleus is highly compartmentalized and chromosomes appear in distinct regions in the nuclear space referred to as “chromosome territories”. A chromosome territory is defined as the space that is occupied by the DNA of a particular chromosome. The chromosome territories are composed of two types of chromatin; heterochromatin and euchromatin. Heterochromatin is highly compacted and transcriptionally inactive and has fewer genes compared to the euchromatin regions, which are transcriptionally active. The chromosome territories are dynamic and permeable structures, accessible for transcription- and regulatory factors (Mahly et al., 2002). Active transcription has been visualized both inside and in the periphery of the chromosome territories (Jackson et al., 1993, Wansink et al., 1993; reviewed by Cremer and Cremer, 2010). The level of compaction of the chromatin is regulated through multiple mechanisms that modulate the interactions between the DNA and the histones, thereby affecting the accessibility of trans-acting factors to the DNA. Two main types of mechanisms regulate the structure of chromatin: ATP-dependent chromatin remodeling and post-translational modifications of histones. Chromatin remodeling factors are multiprotein complexes that modify the structure of the nucleosome. The hBrm protein and its paralog hBrg1 are the ATPase subunits of the SWI/SNF chromatin remodeling complexes in human cells (reviewed by Mohrmann and Verrijzer, 2005). The Brm protein in D. melanogaster (dBrm) is associated with transcribed loci in the polytene chromosomes and plays an important role in ecdysone-dependent transcription regulation (Armstrong et al., 2002). The SWI/SNF complexes participate in the transcriptional regulation of many genes by remodeling nucleosome structure at the promoters (reviewed by Saha et al., 2006). Recent studies in human and Drosophila cells have also shown that Brm regulates alternative processing of pre-mRNA (Batsché et al, 2006; Tyagi et al., 2009).

The nucleolus The nucleolus is the centre for biogenesis. It is the most well characterized subnuclear compartment. The nucleolus is arranged around nucleolar-organizing regions (NORs) which contains repeated ribosomal DNA (rDNA) genes which are transcribed by RNA polymerase I. The number of nucleoli per nucleus varies depending on features as phase in the cell cycle, cell-type as well as metabolic activity. The three major components of the nucleolus are: 1. The Fibrillar center (FC), where the rDNA genes are found; 2. The dense fibrillar center (DFC), here the nascent rRNAs

9 grow and undergo processing; 3. The granular component (GC) is where the final steps of rRNA maturation take place (reviewed by Hernandez-Verdun, 2006) Besides being the center for , the nucleolus has been found involved in functions such as modification of small RNAs, viral replication and cell cycle regulation (reviewed by Boulon et al., 2010). In immuno histochemical studies, the protein Fibrillarin is often used as a marker for the nucleolus. Fibrillarin is found in the NORs and is a marker for newly assembled nucleoli during telophase (Ochs et al., 1985).

Subnuclear compartments

The Cajal body and The Histone locus body Cajal bodies (CBs) were first described by the neuroanatomist Ramon y Cajal more than a hundred years ago. It is a well-studied nuclear organelle that appears as a small spherical structure of 0.5-1µm. It is found in all eukaryotic cells (reviewed by Nizami et al., 2010). Ramon y Cajal named the body “accessory body”, although later it was called Coiled body but since 1999 it is established as Cajal body (Gall, 1999). CBs have a role in the maturation of small RNAs. As an example of that, and snoRNAs localize to CBs before they are translocate to nuclear speckles or nucleoli (Sleeman and Lamond, 1999; Verheggen, 2002). Components that are found in CBs include snoRNA, transcription factors, spliceosomal snRNPs, nucleolar components and 3´-end formation factors (reviewed by Gall, 2000). Additionally the CBs contain non-coding small Cajal body-specific RNA (scaRNAs), a class of snoRNAs that localize specifically to CBs and have been suggested to be involved in the site-specific 2´-O-ribose methylation and pseudouridylation of U2 snRNA (Jády and Kiss, 2001). The protein Coilin is a signature marker for CBs and has been found in mouse, humans (Andrade et al., 1991; Tucker et al., 2000), Xenopus (Tuma et al., 1993), Arabidopsis (Collier et al., 2006) and Drosophila (Liu et al., 2009). Coilin has high similarity in sequence in vertebrates, but coilins from other phylogenetic groups show less sequence similarity along with various molecular weights. Whether there are Coilin orthologues in for example Saccharomyces and Caenorhabditis is still unknown. Work in mouse, Drosophila and Arabidopsis has showed results suggesting that Coilin is needed for CB formation. Additionally the same studies have revealed that either Coilin or CBs are essential for viability (reviewed by Nizami et al., 2010). It is not fully established what functions Coilin has. It has been shown to bind survival motor proteins (SMN), Sm and Lsm proteins. This knowledge has lead to the proposal that Coilin has a function in the assembly and modification of snRNPs (Hebert and Matera, 2000; Hebert et al., 2001; Xu et al., 2005). CBs are dynamic structures, they move, dissociate and reassemble, and exchange contents with the surrounding nucleoplasm. The number of bodies varies in between 1 to 10 depending on cell type and cell cycle. In some cell

10 types CBs are found inside nucleoli but are mostly observed in the nucleoplasm, moreover CBs move inside the nucleolus and in between the nucleoplasm and nucleolus (Boudonck, 1998; 1999; Platani et al., 2000). The Histone locus body (HLB) was initially described as CB as it was observed in Drosophila and human cells. Like CBs, HLBs often contain Coilin in Drosophila, but not in mammalian cells (reviewed by Nizami et al., 2010; Bongiorno-Borbone et al., 2008). The distinction between CBs and HLBs was discovered in Drosophila (Liu et al., 2006). After this discovery, the distinction between the two body species was also identified in human cells (Bongiorno- Borbone et al., 2008; Ghule et al, 2008). HLBs associate with chromosome 2 in Drosophila. I the nucleoplasm HLBs are localized in close proximity to CBs and occasionally also in physical contact. Whether this contact has a functional relevance is still unknown (Liu et al., 2006). As the name indicates most HLB factors are involved in Histone pre-mRNA processing. The intronless Histone pre-mRNA has a 3´extension that has to be removed before export to the cytoplasm (reviewed by Marzluff, 2005). U7 snRNP is the key factor for the cleavage of the 3’ extension. Two examples of other HLB inhabitants are the snRNP proteins Lsm 10 and Lsm 11 (reviewed by Nizami et al., 2010).

The PML body PML nuclear bodies (PML NBs) were identified by electron microscopy 50 years ago as nuclear dense granular bodies and were initially called Nuclear Domain 10 (de The et al., 1960). PML NBs appear as a spherical structure of 0,1-1,0 µm in diameter and are nuclear matrix associated (reviewed by Lallemand-Breitenbach and de Thé, 2010). This matrix is a dynamic, filamentous super structure of the interchromatin part of the nucleus that have been suggested to anchor and regulate numerous nuclear functions such as DNA replication, transcription or epigenetic silencing (reviewed by Nickerson, 2001). PML NBs appear in 5-15 per cell. PML NBs have been implicated in a wide variety of cellular functions among these tumor suppression, viral defense DNA repair and /or transcriptional regulation. PML NBs have been shown to localize to gene rich and transcriptionally active regions of the chromatin, suggesting that PML NBs may have the ability to affect transcription at specific genomic loci (reviewed by Bernardi et al., 2007). The scaffold protein for PML NBs is the PML protein. This protein was discovered in patients suffering form acute promyelocytic leukemia (APL). It has a large number of isoforms with molecular weight spanning 48 to 97 kDa, however the N-terminus is identical in all isoforms (Jensen et al., 2001a). PML is part of the TRIM (Tripartite Motif) family of proteins. TRIM is defined by an RBCC motif containing a conserved RING domain as well as B-box/es and α-helical coiled coiled domains (Borden, 1998). The RBCC domain is crucial for PML dimerization and for assembly into mature PML NBs, a process that is initiated by SUMOylation of the PML protein (reviewed by Lallemand- Breitenbach and de Thé, 2010). PML recruits a large number of proteins. To date 166 interaction partners have been identified. One of the key proteins in

11 the partner recruitment to PML NB is the Small Ubiquitin-like Modifier (SUMO) protein. There are four isoforms of SUMO and all inhabit PML NBs. SUMOylation is a post-translational modification where SUMO is conjugated to a target protein (reviewed by Van Damme et al., 2010). This modification enables the proteins to engage in nuclear transport (Hay 2005; Melchior et al., 2003), transcriptional regulation (Hay 2005; Hamard et al., 2007), apoptosis (Huen and Chen 2008; Li et al., 2006), and protein trafficking (Wilson and Rangasamy 2006). SUMO conjugation involves a cascade of events that are by the activating E1, a conjugating enzyme E2 (or Ubc9) and a protein E3 (Hay 2005). As well as PML, most interaction partners in the PML NBs are SUMOylated and many among these contain SUMO interaction motif (SIM). This fact, together with the presence of (de)-SUMOylation has suggested that PML NBs are nuclear “hotspots for SUMOylation (reviewed by Van Damme et al., 2010). For years the common proposal was that SUMO/SIM were fundamental for PML NB formation, however this view has recently changed when it was shown that specific PML isoforms lacking SIM still generates PML NBs. (Weidtkamp-Peters et al., 2008; reviewed by Lallemand-Breitenbach and de Thé, 2010). This might suggest that SIM/SUMO is not as necessary for PML aggregation as it is for recruitment of proteins to PML NBs. PML NBs are affected by cellular stress, for example, they increase in number upon treatment with interferons and they decrease in number when cells are treated with arsenictrioxide. However, pml-/- mice develop normally despite the inability to form PML NBs (reviewed by Lallemand-Breitenbach and de Thé, 2010).

Nuclear Speckles Nuclear speckles (speckles) are sometimes referred to as SC35 domains or splicing factor compartments. As with the Cajal bodies, it was Ramón y Cajal who first observed this structures and he referred to them as “translucent clumps” (reviewed by Spector and Lamond 2011). However, it was not until the late 1970s and early 80s the connection of speckles to splicing was discovered (Lerner et al., 1981; Perraud et al., 1979; Spector et al., 1983). In mammalian cells speckles appear as irregular dots in a “speckled pattern” in the nucleoplasm. The number of speckles is 25 - 50 per cell and the size is 0.8 -1.8 µm. Speckles contain the pre-mRNA splicing machinery, which involves small nuclear ribonucleoprotein particles (snRNPs), subunits as well as other splicing related factors. The splicing factor proteins SC35 and SF2/ASF are often used as marker proteins for speckles (reviewed by Lamond and Spector 2003; and in Spector and Lamond 2010). It is unclear if any speckles contains genes but they are often localized close to active transcription sites which implies a functional association to (Brown et al., 2008; Huang et al., 1991; Smith et al., 1999; Xing et al., 1995). It is suggested that speckles enable the formation of euchromatic neighborhoods through acting as functional centers that organize active genes. Actively expressed genes can associate at the border of the same speckle (Shopland et al., 2003). Rather than being the actual site of splicing speckles are likely to act as storage, assembly and modification compartments that

12 provide splicing factors to active transcription sites. Experiments have shown that nascent pre-mRNAs are localized outside, but in close proximity to the speckles, in perichromatin fibrils (PFs). PFs that are situated close to the speckles are difficult to distinguish from speckles by fluorescence microscopy, which has caused some controversy in the discussion whether speckles are directly involved in splicing or not (reviewed by Spector and Lamond 2011). Speckles are dynamic structures that vary in size and number according to changes in gene expression levels. For instance, speckles become round and increase in size when transcription is inhibited (Melcak et al., 2000; Spector et al., 1991; Spector et al., 1983) and the level of splicing factors decrease in speckles if transcription is put to an intense level by viral infection (Bridge et al., 1995; Jiménez-García et al., 1993) or if expression of intron containing genes increase (Huang et al., 1996; Misteli et al., 1997).

Paraspeckles Paraspeckles were discovered as late as in 2002. The discovery of the protein PSPC1 revealed the presence of this novel substructure and it is now the prominent marker protein for paraspeckles (Andersen et al., 2002; Fox et al., 2002). These nuclear compartments are seen as irregular, sausage-like shaped structures in number of 5 – 20 per nucleus. The size varies, but it range in between 0.5 - 1µm in diameter. Paraspeckles have been observed in the nuclei of all mammalian cells and tissues, human cell lines, primary cell lines and embryonic fibroblasts examined, with the exception of human embryonic stem cells (reviewed by Fox and Lamond 2010). A relatively low amount of proteins for example PSPC1 (required for paraspeckle formation in HeLa cells), p54nrb and PSF assemble in paraspeckles These three proteins are all part of the DBHS family of proteins that normally cycle between the nucleoplasm, paraspeckles, and the nucleolus but accumulate within perinucleolar cap structures when Pol II transcription is inhibited (Fox et al., 2002; Prasanth et al., 2005; Shav-Tal et al., 2005). Paraspeckles do not overlap with sites of active transcription although the majority of the proteins in paraspeckles are involved in RNA polymerase II transcription and/or RNA processing. In addition to the protein content, paraspeckles also contain at least two RNA species: NEAT1 (Clemson et al., 2009; Sunwoo et al., 2009; Sasaki et al., 2009) and Ctn (Prsanth et al., 2005). NEAT1 is required for the maintenance and structure of the paraspeckle assembly. Ctn is regulated in paraspeckles and gives paraspeckles a role in RNA retention by preventing A to I hyper edited RNA from leaving the nucleus as part of control of gene expression of the mouse CAT2. CAT2 is a protein needed for wound healing and defense against infection (reviewed by Fox and Lamond 2010).

Orphan bodies Orphan body is a novel classification of nuclear bodies that are less established than the relatively well characterized nuclear bodies described above. One does not find the orphan bodies in all cell types and generally these are dynamic structures that are sensitive to stages in cell cycle and to environmental

13 changes. So far, the cluster of nuclear bodies included in this group are the OPT domain, polycomb body, clastosome, Sam68 body, cleavage body and SUMO body (reviewed by Carmo-Fonseca et al. 2010).

Oct1/PTF/transcription (OPT) domain. OPT domains appear as round structures of 1-1.5 µm in diameter 1-3 per cell. This domain is enriched in transcription factors Oct1 and PTF (Pombo et al., 1998; Grande et al., 1997). OPT domains assemble during G1 phase in the cell cycle and disappear during S phase. OPT domains are transcription dependent though they disrupt upon DRB treatment (Pombo et al., 1998).

Clastosome. degrade short-lived proteins involved in , cell cycle and gene transcription. Additionally the degrades erroneous proteins and plays a role in the adaptive immune response. Proteins destined for degradation are ubiquinated by a chain of several copies of the small protein ubiquitin as flag for degradation. Proteins can be degraded by the ubiquitin-proteasome system in both cytoplasm and nucleus. Now and then proteasomes accumulate in discrete ring-shaped nuclear bodies together with ubiquitin and the two degradation targets c-Jun and c-Fos. The proteasome-containing bodies, called clastosomes, alter upon stimulation. For example clastosomes increase in number by induced expression of c-fos protein and disappear by proteasome inhibition (Lafarga et al., 2002 and reviewed by Carmo-Fonseca 2010). Furthermore the PML protein co localizes with ubiquitin and proteasome in nuclear bodies. It is not possible to differentiate mature PML bodies from clastosomes by electron microscopy though both body types have the typical doughnut shape and size. It has been suggested that SUMOylated PML recruits polyubiquitination and proteasomal degradation, which are supposed to support the maturation of PML bodies (Lallemand- Breitenbach et al., 2001). Another proposal is that SUMO-modified PML protein is recruited to clastosomes. Clastosomes can assemble without PML protein (Lafarga et al, 2002), moreover the dominant (in some cell types up to 90%) part of PML protein has a diffuse distribution in the nucleoplasm and not located nuclear bodies (Lallemand-Breitenbach et al., 2001).

Polycomb body (PcG body). Polycomb group (PcG) proteins were initially discovered as factors responsible for keeping Hox/homeotic genes repressed in Drosophila development (Buchenau et al., 1998). PcG proteins are now observed in other species and have been identified as well-conserved and essential proteins that target specific DNA regions and repress transcription of, for instance, developmental genes (Schuettengruber et al., 2007). PcG proteins are found in repressive complexes along with other interacting factors. In mammals and Drosophila polycomb repressive complex 1 (PRC1) includes PcG protein, HPH1/HPH2, Bmi1, HPC2 and RING1 proteins are colocalized in PcG bodies PcG bodies are of round or irregular shape, 0.3 -1 µm in diameter and in number 12 to 16 per cell. (Buchenau et al., 1998; Saurin et al., 1998).

14 As in the case of PML bodies, PcG bodies are connected to the SUMOylation pathway; both are enriched with the SUMO-conjugating enzyme Ubc9. It is suggested that both these nuclear compartments are centers for SUMOylation. It is suggested that PcG bodies are repression centers for repressed proteins, that even though a protein has been deSUMOylated the protein stays repressed inside the repression center in form of a PcG body (Hay 2005).

Sam68 body. Sam68, SLM-1 and SLM-2 proteins are all part of the signal transduction and activation of RNA (STAR) family of proteins. These RNA- binding proteins assemble in nuclear bodies that disassemble during mitosis and upon transcriptional inhibition. The bodies appear as 0.3 – 1.0 µm in diameter in between 2 and 5 bodies per cell (reviewed by Carmo-Fonseca et al. 2010). It is believed that Sam68 bodies contain RNA since electron microscopic experiments have revealed the presence of nucleic acids. They have at times been observed close to Cajal bodies and nucleolus (Chen et al., 1999). It has also been reported that Sam68 is SUMO modified (Babic et al., 2006).

Cleavage body. This nuclear compartment prominently contains the cleavage stimulation factor (CstF) and the cleavage and polyadenylation specificity factor (CPSF) as well as other factors such as the DEAD box protein DDX1. Cleavage bodies are involved in the processing of the 3´-end of almost all mRNAs. Cleavage bodies are primarily seen during S phase and are not sensitive to transcription inhibition nor contain RNA. The number per cell ranges between 1 and 4 and the diameter is 0.2-1.0 µm (Schul et al., 1996; Bléoo et al., 2001; Li et al., 2006; Spector 2006)

SUMO body (SNB). This novel nuclear body is enriched in active conjugating SUMO-1 and Ubc9. The number of bodies per cell span 1 to 3 and the size in between 1 to 3 µm. Additionally, SNBs assemble the transcriptional regulators CBP, CREB, and c-Jun. SNB was discovered in the mouse UR61 cell line where it was suggested to play a role in the control of the nuclear concentration of transcription regulators that are involved in neuroprotection and survival of these kind of neuron-like cells (Navascués et al., 2007).

Expression of protein-coding genes in eukaryotic cells

Expression of protein-coding genes in the eukaryotic cell involves a large number of essential events that result in the formation of a messenger RNA (mRNA) mature and ready to be efficiently exported to the cytoplasm. These different events start with the initiation, elongation, and termination of transcription by the RNA polymerase II (Pol II). The maturation of the mRNA involves loading of a number of RNA binding proteins (hnRNP proteins) onto the nascent RNA and the assembly of a ribonucleoprotein (RNP) complex.

15 Before the mRNP is mature and ready to be exported from the nucleus to the cytoplasm in order to be translated, it has to go through the proper processing. This processing includes capping of the 5’ end, splicing of introns, cleavage and polyadenylation of the 3´end, followed by the release from the transcription site.

Co-transcriptional mRNA processing

It is well established that most of the events mentioned above occur co- transcriptionally. This means that they take place during ongoing RNA synthesis, while the transcript is still physically connected to the RNA polymerase II. In recent years, it has also become clear that the different processing events are interconnected to a large extent, and it has been suggested that this enhances the efficiency and the specificity of the gene expression. However, not all processing events are necessarily coupled to transcription itself (reviewed by Maniatis and Reed, 2002).

CTD The C-terminal domain (CTD) of the large subunit of Pol II plays a key role in coordinating the coupling of pre-mRNA processing and transcription. The CTD consists of heptad repeats (YSPTSPS), 52 in human and 27 in budding yeast. Phosphorylation and dephosphorylation of the CTD of Pol II are important events during the transcription process. The phosphorylation pattern of the CTD changes during transcription. It is the hypophosphorylated form of Pol II that binds the pre-initiation complex and later, as Pol-II goes into elongation, the CTD becomes phosphorylated (Lu et al., 1991; Chesnut et al., 1992). It is primarily the serine 5 residues that are phosphorylated at the promoter region, and the serine 2 is only seen phosphorylated in coding regions (Komarnitsky et al., 2000). Deletions of domains in the CTD have revealed the importance of the CTD in the recruitment of various factors via phosphorylation of different residues during capping (McCracken et al., 1997a; Cho et al., 1997), splicing and polyadenylation (McCracken et al., 1997b). In humans certain parts of the CTD appear to have specialized functions and interact with factors specific for the different processing events (Fong and Bentley, 2001).

5´end capping Transcripts transcribed by Pol II are capped at their 5´ends and this process is facilitated by the binding of capping enzymes to Pol II (Shatkin and Manley, 2000). Capping is the first processing step and occurs during the transition from initiation to elongation, when the transcript is only 20-40 nucleotides long. During the initiation, serine 5 in the CTD heptad becomes phosphorylated, which leads to the dissociation of initiation factors from the CTD and results in the association of the capping machinery with the CTD. Dephosphorylation of serine 5 results, at later stages, in the release of the capping machinery.

16 Capping is a three-step process that adds a GMP cap structure to the 5´end of all mRNAs. It begins with the hydrolysis of the triphosphate of the first nucleotide of the pre-mRNA into a diphosphate by an RNA 5´triphosphatase. In the second step, a GMT moiety from GTP is ligated to the first nucleotide of the pre-mRNA. The fusion is catalyzed by a guanylyltransferase. In the final step of the capping, a methyltransferase methylates the N7 position of the GMT. The cap structure is then recognized by the cap binding complex, a complex of two proteins: CBP80 and CBP20 (Ohno et al., 1990; Izaurralde et al., 1994; Izaurralde et al., 1995). The binding of the CBC stabilizes the pre- mRNA, protecting it from degradation by 5´-3´ (reviewed by Decker and Parker, 1994).

Splicing To generate a functional transcript, the introns that interrupt the coding sequence in the pre-mRNA have to be spliced out. The transcript itself contains consensus sequences that make the splicing reactions possible. The 5´exon- intron splice-site consists of the consensus sequence AG/GURAGU (R, Purine; Y, Pyrimidine). The 3´splice-site is recognized as YAG/RNNN. A branch point with a highly conserved adenosine followed by a pyrimidine-rich tract referred to as “polypyrimidine tract” is found upstream of the 3´ splice-site. The splicing reaction is a two-step reaction carried out by the spliceosome, a complex of numerous components that are assembled in a conserved stepwise order (reviewed by Krämer, 1996; Reed, 2000). The splicing reaction starts with the cleavage at the 5´-splice site. This cleavage is promoted by the 2´OH of the branch point adenosine and results in a free 5´exon and a lariat molecule that consists of the intron and the 3´exon. In the second step, the 3´OH of the upstream 5´exon loops back and attacks the 3´exon edge and creates a lariat structure containing the intron sequence. This results in the ligation of the two exons whereas the lariat is cleaved and released. The two exons are joined, which completes the splicing reaction. The key players that mediate these two reactions are five small nuclear RNAs (snRNAs): U1, U2, U4, U5 and U6 snRNAs. Each of these RNAs comes together in an UsnRNP complex. The snRNPs, together with other proteins, such as the SR proteins ASF/SF2 and SC35, and others, form the spliceosome, which brings the 3´splice site close to the 5´site and the branch point. Once the three key positions are brought together the splicing reaction can be preformed (reviewed by Kramer, 1996). Splicing occurs to a great deal co-transcriptionally (Bauren and Wieslander, 1994). It has been observed that splicing factors co-localize with actively transcribing Pol II (Osheim et al., 1985; Kiseleva et al., 1994; Misteli and Spector, 1999). It has also been reported that CTD phosphorylation enhances an early step in splicing and is required for co-transcriptional splicing and poly (A) site cleavage (Hirose, 1999; Zeng, 2000). During the splicing reaction, a multi- is deposited in a fixed position on the spliced mRNA. This complex, located 25 bp upstream of the newly formed exon-exon junction is called the Exon-exon Junction Complex (EJC) (LeHir et al., 2000a; 2000b; 2001). The EJC comprises the four proteins

17 MAGOH, Y14, MLN51 and eIF4AIII (LeHir et al. 2000 b; Kataoka et al., 2001; Kim et al., 2001; Chia et al., 2004; Degot et al., 2004). Other proteins that also associate with the EJC are splicing factors (Srm160, pinin and RNPS1), mRNA export factors (REF/Aly and Tap-p15) and factors that are involved in cytoplasmic mRNA surveillance (UPF3 and UPF2) (reviewed by Tange et al., 2004).

3´end formation and termination The formation of the 3’end of the mRNA involves two reactions: cleavage and polyadenylation at the 3´end of the transcript. These two reactions are coupled and lead to a poly (A) tail of 200-300 adenosines in mammals, and 70-90 nucleotides in yeast. The cleavage site is located between the conserved hexamer sequence AAUAAA and the downstream sequence element that consists of U or GU-rich motif. Two different protein complexes recognize the cleavage site: cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulatory factor (CstF). CPSF binds the AAUAAA sequence and CstF binds the downstream sequence element (DSE). The binding of CstF is likely to increase the affinity of CPSF to its and the other way around. Cleavage is preformed by the cleavage factors I and II (CF1 and CFII). Besides the poly (A) addition reaction, the poly (A) polymerase (PAP) is also important for cleavage through its interaction with CPSF. PAP and CPSF direct together the poly (A) addition. The processivity of PAP is increased by the nuclear poly (A)-binding protein PABP II (reviewed by Colgan and Manley, 1997). The cleavage of the pre-mRNA results in two products. The product that is supposed to become mRNA is stabilized by the poly(A)-tail while the downstream product is readily degraded by exonucleases. As in the case of capping and splicing, the process of termination and 3´end processing need factors that interact with the CTD. In vitro experiments have shown that a number of 3´end processing factors associate with CTD phosphorylated at ser 2 (Barilla et al., 2001; Licatalosi et al., 2002). The CTD has also been shown to increase the efficiency of polyadenylation (Fong & Bentley, 2001). Direct interactions between the CTD and both CPSF and CstF have been observed (McCracken et al., 1997b; Fong et al., 2001). The transcription process ends with the termination. Termination allows the release of the transcript from the transcription site and also the release of Pol II from the template. Pol II is able to proceed transcribing up to 2 kb after the cleavage site. Cleavage and polyadenylation are events that are distinct from termination, although cleavage is necessary for termination and termination depends on the poly (A) signal (reviewed by Sims et al., 2004).

Export and retention The export of mRNA to the cytoplasm is a signal-mediated pathway. mRNA export requires dynamic rearrangements of protein-protein and protein-RNA interactions to form an export-competent mRNP complex. The rearrangements result in the dissociation of retention factors and the assembly of signals that are necessary for the interactions with the export machinery. A large number of

18 factors are responsible for export of the mRNA, including mRNA binding proteins, nucleoporins and ATPase/RNA . The key players in the yeast and metazoan mRNA export are the conserved nuclear export factors (or NXF) Mex67p in yeast and TAP/NXF1 in metazoans (Herold et al., 2000). Mex67p and TAP/NXF1 form heterodimers with Mtr2p and p15/Nxt1, respectively (Katahira et al., 1999; Santos-Rosa et al., 1998). Mtr2p and p15/Nxt1 are required for the binding of Mex67p and TAP/NFX1 to the nuclear pore complex or NPC (Fribourg et al., 2001; Levesque et al., 2001; Strasser et al., 2000a; Wiegand et al., 2002). Mex67p and TAP/NFX1 themselves have low affinity for RNA (Katahira et al., 1999; Santos-Rosa et al., 1998), and in most cases the RNA interaction is mediated by the hnRNP- like adaptor proteins Yra1p in yeast, and Aly in higher eukaryotes. These proteins belong to the REF (RNA and Export Factor binding proteins) family of hnRNP-like proteins. The adaptor proteins bind to RNA and directly interact with Mex67p or TAP/NXF1. For instance, in S. cerevisiae Yra1/REF recruits Mex67p to the mRNP (Zenklusen et al., 2001) and is essential for mRNA export (Strasser et al., 2000b; Stutz et al., 2000). In Xenopus oocytes, Aly/REF was shown to promote mRNA export, and mRNA export could be blocked by antibodies against Aly (Rodrigues et al., 2001). Another player in mRNA export is the THO complex. In yeast, the THO complex comprises four subunits -Hpr1p, Mft1p, Tho2p and Thp1p- and it interacts with Pol II and with the emerging RNA at the site of transcription during elongation (Chang et al. 1999; Strasser et al. 2002a; Zenklusen et al. 2002). Mutating any of the THO components in yeast leads to phenotypes like those in transcription impairment, and also leads to defects in mRNA export (Chavez et al., 2000; Rondon et al., 2003; Schneiter et al., 1999; Strasser et al. 2002). The yeast THO complex associates with two other factors, Sub2p and Yra1p, which have their counterparts in the human splicing factor UAP56 and export factor Aly/REF, respectively. Sub2 is an ATPase/RNA helicases that recruits adaptor proteins to the mRNP. THO, Sub2p and Yra1p constitute the TREX complex, for TRanscription and EXport complex (Strasser et al., 2002). In yeast THO/Sub2 mutants show dotted pattern of accumulated nascent transcripts from the HSP104 gene (Libri et al., 2002). The same pattern was observed in mammalian cells with THO/TREX depletion, where nascent transcripts from the HSP70 gene were accumulated presumably at or near the site of transcription (Mandel et al., 2008). Beside the THO/TREX dependent recruitment of Mex67, an alternative recruitment site for Mex67 is provided by the SR-like protein Npl1. Npl1 works as an adaptor of Mex67 and RNA (Gilbert et al., 2004). This type of interaction has not been observed for other shuttling mRNA-binding proteins in yeast, such as Nab2p, Hrb1p and Hpr1p, but their shuttling is RNA synthesis- dependent and they are needed for export. This suggests that these factors contribute to the export in a passive manner. Their role could be to serve as marks for proper processing and packaging of the mRNP complex (reviewed by Zenklusen et al., 2001). mRNP export also requires the DEAD box protein Dbp5 (Snay -Hodge et al., 1998). This binds to the cytoplasmic filaments of the NPC (Hodge et

19 al., 1999) and it has been suggested that its ATPase activity is used to pull the mRNP through the channel of the NPC (Hodge et al., 1999; Schmitt et al., 1999; Zhao et al., 2002). When the Mex67p-Mtr2p and TAP/NXF1-p15/Nxt1 heterodimers are associated with the mature mRNP, they promote the translocation of the mRNP complex through the NPC by interacting with FG-nucleoporins positioned in the central channel of the NPC (Conti et al., 2001).

Coupling between the processing events

As mentioned earlier, the mRNA processing events are interconnected and affect each other to a large extent. A selection of examples of coupling between the events will be presented here. Several reports have gathered to the conclusion that transcription and capping are connected. Transcription complexes are kept at the promoter until capping takes place then the polymerase switches into an elongating form (reviewed by Hocine et al., 2010). Capping and splicing are connected. Early experiments in Xenopus showed that depletion of the CBC results in inhibition of splicing. This is due to the fact that the CBC increases the interaction between the U1 snRNP and the 5´splice site (reviewed by Lewis and Izaurralde, 1997). The same type of interaction has also been demonstrated in studies in S. cerevisiae (Shatkin and Manley, 2000). On the other hand, the CBC also has a stabilizing effect on the polyadenylation complex and enhances the cleavage reaction (Flaherty et al., 1997). It has been shown in yeast mutants that impairing mRNA export affected transcription negatively (Luna, et al., 2005). Luna and co-workers showed that the THO and TREX complexes have an important role in the mRNP formation, concluding that transcription elongation and termination, mRNP biogenesis and export are highly interdependent (Luna, et al., 2005). It has ben shown that THO/TREX components in mammalian cells are recruited to the CBC during the splicing reaction (Cheng et al., 2006; Nojima et al., 2007). ChIP experiments in yeast have revealed that Hrp1, Sub2 and Yra1 are recruited co- transcriptionally during elongation at a similar time (Zenklusen et al., 2002). Hrp1 was shown to be required for efficient recruitment of Yra1 and Sub2. Depleting or mutating any of these factors leads to the decrease of mRNA levels, which suggested that improper loading of mRNA export factors result in mRNP instability (Zenklusen et al., 2002). Although splicing and export are distinct processes located at spatially different locations in the nucleus, these events are also coupled. It has been shown in Xenopus oocytes that pre-mRNA splicing is coupled to mRNA export. Injection experiments showed that spliced mRNA was exported to a higher rate than the corresponding synthetic non-intron containing transcripts (Luo et al., 2001). In higher eukaryotes, mRNAs generated from splicing become associated with a specific set of proteins or EJC as explained above (Le Hir et al., 2000). Both the export factors Aly/REF and UAP56 are recruited to the EJC during the splicing reaction (Gatfield et al. 2001; Le Hir et al. 2000).

20 UAP56 is in itself a splicing factor and it has been proposed that UAP56 recruits Aly/REF to the spliced mRNA (Strässer and Hurt, 2001). UAP56 and TAP/NXF1 have been shown to be necessary for the export of spliced mRNA and of mRNA derived from intron-less genes (Gatfield et al., 2002; Herold et al., 2003; Longman et al., 2003). In both yeast and higher eukaryotes, mRNA export is dependent on the proper 3´end formation (Eckner et al., 1991; Huang and Carmichael, 1996). It has been observed that in stable murine erythroleukemia cell lines expressing mutant and wild-type forms of human beta-globin, the beta-globin mRNAs with defects in 3´end cleavage are retained at the site of transcription. Retention is also observed in mutants that are defective in splicing. This was shown by using the method transcript-specific RNA-Fluorescent In Situ Hybridization (RNA-FISH) (Custodio et al., 1999). The end of the terminal exon of a transcript is defined by the poly (A) signal. Both splicing and polyadenylation enhance each other on both sides of the exon (Niwa et al., 1992) by the interaction of U2AF 65 and the c-terminal domain of poly (A) polymerase (Vagner et al., 2000). There are also examples of alternative polyadenylation, where weak poly (A) sites are repressed or enhanced by the upstream splicing machinery (Takagaki et al., 1996; Takagaki and Manley, 1998). Recent data has demonstrated that transcription termination and initiation are also coupled events (Mapendano et al., 2010). This study presents evidence that 3´end formation may stimulate initiation of transcription. Disrupted 3´end formation and transcription termination in HEK293 cell line caused a decrease in gene-associated RNA pol II as well as promoter–attached TBP and TFIIB. This data lead to the proposal that transcription factors in normal cells are re- cycled. At the same time this mechanism serves as an effective way to shut down transcription if transcription termination fails (Mapendano et al., 2010). It has been shown in yeas that genes may form loops that connect the 5´and 3´ends of a gene (O´Sullivan et al., 2004). Moreover gene loops have been shown to associate to the NPC (Rougmaille et al., 2008). This mechanism may provide a physical network that enhances the efficiency of transcription.

mRNA surveillance

Co-transcriptional coupling between gene expression events might provide a surveillance system where the interdependence between the many different factors and actions leads to the recognition of errors at a subsequent stage. The term mRNA surveillance includes several types of mechanisms that lead to degradation of defective transcripts and selective expression of correct ones. The surveillance pathways monitor many of the steps during gene expression, from transcription to export of the mature mRNA. In general there are two different degradative mechanisms by which erroneous and bulk mRNAs are degraded in both the cytoplasm and the nucleus. The enzymes responsible for the decay degrade in either 5´- 3´ or 3´- 5´ direction, depending on the RNA

21 substrate and on the enzyme (Libri et al., 2002; Kufel et al., 2004; Bousquet- Antonelli et al., 2000). mRNA surveillance pathways include two steps: the first one is the recognition of the erroneous transcript, and the second one its degradation. Recently discovered surveillance mechanisms also involve transcriptional down regulation (Damgaard et al., 2008; Paper II: Eberle et al., 2010). The best understood surveillance pathway is Nonsense-Mediated Decay or NMD that occurs in the cytoplasm. NMD is a posttranscriptional pathway that identifies and degrades mRNAs that contain a premature translation termination codon. In mammals, degradation of the mRNA by NMD, generally takes place if translation terminates about 50 to 55 nucleotides upstream of an exon-exon junction with the EJC present 20 -25 nt upstream of the exon-exon junction. The EJC serves as a mark for decay when the translating ribosome has failed to remove the factors inhibiting the activities of the EJC (Gonzales et al., 2001; Le Hir et al., 2001). If the mammalian mRNA does not carry a exon-exon junction the NMD will not occur, however this is not the case for most other eukaryotes as S. cerevisiae, S. pombe, C. elegans, D. melanogaster and plants in which other elements contribute to define a codon as premature (Maquat, 2004). For instance, the distance between the incorrect stop codon and the poly (A) tail is predicted to be the target for premature stop codon definition in yeast (Amrani et al., 2004). In mammals, the proteins Upf2, Upf3/Upf3X and presumably transiently also Upf1, associate with the EJC and trigger NMD. They recognize the premature termination codon in conjugation with the translational release factors eRF1 and eRF3 after an initial round of translation (Maquat, 2005). NMD in mammalian cells include both 5´-3´ and 3´- 5´-exonucleolytic degradation. It has been shown in mammalian cells that Upf1, Upf2 and Upf3/X co- immunopurify with the decapping enzyme Dcp2, the 5´-3´ Xrn1, and the exosome components PM/scl100, Rrp4, Rrp41 and PARN (Lejune et al., 2003). Data from this study conclude that NMD exonucleolytically degrades mRNA from the 5´ to 3´ end by the Rat1 in S. cerevisiae (and Xrn1 in mammals) upon decapping and after deadenylation from the 3´to 5´end by the exosome. In Drosophila, however, NMD is usually initiated by an endonucleolytic cleavage (Gatfield and Izaurralde, 2004). Besides having a role in NMD, the exosome mediates another branch of mRNA surveillance, where it degrades mRNAs with defects in 3´-end formation (reviewed by Saguez et al., 2005). The role of the exosome in this process has been well established by genetic studies in S. cerevisiae but the mechanism is not well understood (see below). Another type of mRNA surveillance mechanism discovered in S. cerevisiae was that mediated by the Mlp1/2 proteins, orthologs of the mammalian Tpr and the Mtor in insects. Again based on genetic studies, it has been shown that Mlp proteins are involved in the recognition and nuclear retention of intron- containing mRNAs at the nuclear envelope (Galy et al., 2004).

22 The exosome

The exosome is one of the most important players when it comes to maintaining the correct RNA levels in the cell, in both the cytoplasm and the nucleus. The exosome is an evolutionarily conserved multiprotein complex that is found in both archeabacteria and eukaryotes (reviewed by Lykke–Andersen et al., 2009).

Exosome complexes in bacteria and archea

There are exosome-related RNA exonuclease complexes in both bacteria and archea. In E. coli, mRNA degradation is mediated by the combined action of endo- and exonucleases. The 3´- 5´degradation pathway is handled by the (Erhetsmann et al., 1992; Carpousis et al., 1994). The major participants of this complex are RNaseE, polynucleotide phosphorylase (PNPase) and the DEAD-box RNA helicase B (RhIB) (Py et al., 1996; Miczak et al., 1996; Blum et al., 1996). The PNPase consists of two RNase PH domains, one S1 domain and one KH domain in a single polypeptide. The PNPase forms a hexameric ring of three homodimers, where the three S1 and KH domains from each subunit are arranged on the same side of the ring. This structure is found in eubacteria, mitochondria and (Symmons et al., 2000, and reviewed by Symmons et al., 2002). The archeal exosome is the best mapped of all exosome complexes and it shows great similarities with the bacterial PNPase, but with the exception that the different domains are found in different subunits (Symmons et al., 2000). The archeal exosome has been described as a macromolecular cage (reviewed by Buttner et al., 2006). It has a ring-like architecture with a hollow central channel. This channel, or processing chamber, inhabits three phosphorolytic active sites. For the exosome to be able to degrade its substrates, the single stranded, unfolded RNA substrate has to be pulled through the central pore to reach the active sites of degradation. The archeal exosome subunits Rrp41 and Rrp42, which contain RNase PH- like domains, alternate in the ring structure that consists of six subunits and is 90 Å wide and 70 Å thick (Lorentzen et al., 2005). A second ring structure that binds the Rrp41: Rrp42 ring consists of three copies of Rrp4 and/or Csl4, two factors that carry the S1/KH RNA-binding domains. This second ring creates the entry of the complex (Lorentzen et al., 2005; Buttner et al., 2005). The S1 domains are found towards the centre of the complex and create the so-called S1 pore. The KH domain of Rrp4 and the zinc-ribbon domain of Csl4 are located at the periphery of the S1 ring (Buttner et al., 2005). The location of the three different nucleic acid binding domains of the Rrp4/Csl4 ring presents a large area for recruiting RNA substrates (reviewed by Buttner et al., 2006). It has been shown that functional non-stoichiometric sub complexes, with different substrate specificities of the exosome are common in Archea (Buttner et al., 2005; Evguenieva-Hackenberg et al., 2008; Lorentzen and Conti, 2008; Roppelt et al., 2010).

23 The active sites of degradation are located in the Rrp41 subunit of the archeal exosome, located at the end of an internal groove of about 20Å. These active sites are not accessible unless the RNA substrates pass through the central channel of the exosome. Rrp41 is inactive by itself, and requires Rrp42 to contribute to the structure of the (Lorentzen et al., 2005). The entry of the central channel (e.g. the S1 pore) is a narrow pore of 8-10 Å, a size that accommodates only single-stranded RNA substrates and prevents uncontrolled RNA decay. The size of the exit is considerably larger; about 20-25 Å.

Figure 2. The exosome structure is conserved. The top panel shows the archeal and eukaryotic exosomes from above. Both have a common overall structure with a ring of six RNase PH domains and a top cap composed of three subunits. Three copies of two proteins constitute the archeal PH ring and the cap consists of either one of two proteins. The eukaryotic PH ring is

composed of six individual proteins and three different cap proteins. The bottom panel shows a “cut away” view from the side of both complexes to illustrate the RNA inside the channel and point out the sites of degradation. In archea RNA enters the PH ring via the cap and RNA degradation occurs inside the central channel. In eukaryotes (yeast and human) the ribonucleic catalysis is carried out by a tenth subunit -Dis3/Rrp44- that binds to the opposite side to the cap proteins. It has not been proved that RNA passes through the central channel before catalysis in eukaryotes (Figure adapted from Tsanova and van Hoof, 2010).

24

The exosome in eukaryotes

Since the discovery of the yeast in S. cerevisiae (Mitchell et al., 1997; Allmang et al., 1999b), this conserved complex has been identified in several other eukaryotic species including plants (Chekanova et al., 2000; reviewed by Lykke-Andersen et al., 2009), flies (Andrulis et al., 2002) and humans (Allmang et al., 1999b; Brouwer et al., 2000). The exact composition of the eukaryotic exosome is becoming clearer, but is to some extent in the dark. It is easy to conclude that the eukaryotic exosome is more complex and diversified in structure and composition than the archeal one, because the number of distinct subunits of the eukaryotic exosome is larger than in archea. In the yeast complex, the archeal Rrp41 is represented based on sequence comparison, by three subunits with RNase PH domains: Rrp41, Rrp46 and Mtr3. Rrp42 has three orthologs: Rrp42, Rrp43 and Rrp45. In spite of the higher number of different components, it has been suggested that the eukaryotic exosome resembles the archeal exosome in structure (Aloy, et al., 2002; Hernandez et al., 2006). The current models for the eukaryotic exosome propose the existence of a core formed by alternating Rrp41-like and Rrp42-like proteins in a hexameric ring, as is the case in archea (Esteves et al., 2003; Lehner et al., 2004; Raijmakers et al., 2002; Lorentzen et al., 2005). The S1/KH proteins Rrp4 and Csl4 are represented in eukaryotes by the yeast Rrp4, Rrp40 and Csl4 (Mitchell et al., 1997; Allmang et al., 1999b; Chekanova et al., 2002). Despite the high level of conservation in between archeal and eukaryotic exosome complexes, the eukaryotic exosome (with some exceptions) has lost its active sites for degradation in the core channel (Dziembowski et al., 2007; Liu et al., 2006). The exonucleolytic activity of the eukaryotic exosome is supplied by two hydrolytic 3´- 5´, Rrp6 and Dis3/Rrp44 that interact with the core (Liu et al., 2006; reviewed by Tomecki et al., 2010). The eukaryotic exosome is found in both cytoplasm and nucleus. The nuclear and cytoplasmic forms of the exosome differ slightly, but they share ten common subunits. The nuclear exosome in yeast is characterized by the presence of Rrp6p and Rrp47, whereas the cytoplasmic exosome contains the GTPase Ski7 (Allmang et al., 1999b; Mitchell and Tollervey, 2003; Synowsky et al., 2009). The Drosophila exosome was purified by immunoprecipitation (Andrulis et al., 2002; Graham et al., 2006). The Drosophila exosome isolated by Andrulis and coworkers lacked Rrp43 and Rrp45, which are part of the yeast exosome, although there is a Drosophila homologue for Rrp45 (Andrulis et al., 2002). The Andrulis lab has recently carried out an immuno-fluorescence study of the Drosophila exosome in S2 cells using both epitope-tagged subunits and antibodies against endogenous factors in order to study the interaction and localization of the different exosome subunits (Graham et al., 2006). These studies revealed that exosome subunits are assembled in sub compartments and led to the suggestion that different exosome subunits exist in sub-complexes at distinct locations in vivo. Graham et al. (2006) could conclude that the exosome in Drosophila is not a unique complex with constant composition, and they proposed instead the existence of multiple exosome variants with

25 specialized functions (Graham et al., 2006). Other reports in vivo and in vitro have shown that subsets of exosome subunits can perform RNA metabolic functions (Schneider et al., 2007; Callahan and Butler, 2008; 2010). Considering the reports mentioned above, D.L. Kiss and E.D. Andrulis have recently presented a novel revised view regarding the exosome complex that they refer to as the exozyme model (Kiss and Andrulis, 2011). Exozymes are suggested to be composed of exosome subunits either alone or in a combination of exosome subunits and cofactors. The molecular masses of the exozymes vary in size depending on the subunit composition. It is also proposed that exozyme complexes function independently of other exozymes with different RNA substrate specificities, or share the same RNAs, but during different steps of processing. Furthermore, the smaller exozymes may dissociate from the core exosome or stay in a dynamic association with the core or other exosome subunits (Kiss and Andrulis 2011).

The catalytic subunits of the exosome

In S. cerevisiae, the 110 kDa Dis3 (Rrp44) protein associates with the core exosome in both cytoplasm and nucleus (Lorentzen et al., 2008). Dis3 (Rrp44) is essential for the activity of the exosome in yeast (Dziembowski et al., 2007). It has been shown that Dis3 (Rrp44) exhibits endonucleolytic as well as exonucleolytic activities through the functional PilT N-terminus (PIN) domain (Leberton et al., 2008; Schaeffer et al., 2009). In D. melanogaster Dis3 interacts with the core exosome through interaction between the Dis3 N- terminus and the Rrp41 and Rrp45 subunits (Bonneau et al., 2009; Graham et al., 2006; 2009; Mamolen et al., 2010). A recent report has revealed a previously unknown association of a Dis3 (Rrp44) homologue to the human core exosome (Staals et al., 2010). The novel human Dis3-like (Dis3L1) was co-immunoprecipitated to the cytoplasmic exosome and was shown to exhibit exoribonuclease activity (Staals et al., 2010). In yeast the Rrp6 exoribonuclease exclusively localizes to the nucleus and the nuclear core exosome (Allmang et al., 1999b). In human cells PM/Scl-100 is localized to both nucleus and cytoplasm (Brouwer et al., 2001; Lejune et al., 2003), and the same has been observed in Drosophila and trypanosome (Graham et al., 2006; Haile et al., 2007). Rrp6 is not essential for viability in yeast (Briggs et al., 1998). Depletions of Rrp6 in different species have shown phenotype changes that are distinct from those in cells with other depleted exosome components, and it has been suggested that Rrp6 has additional exosome-independent functions in the cell (van Dijk et al., 2007; Callahan and Butler 2008; Graham et al., 2009). As an example, Rrp6 is able to carry out 3´end processing of 5.8 S rRNA and snoRNA without functional exosome in human cells (Callahan and Butler 2008), and has a role in cell cycle and mitotic progression in Drosophila (Graham et al., 2009). The precise location of Rrp6 in relation to the core exosome is not known. It has been proposed that Rrp6 interacts with the top part of the exosome at the side of the hexameric ring (Cristodero et al., 2008).

26 Fractionation studies in yeast have concluded that the cytoplasmic core exosome is a stable complex that remains detectable at Mg2+ concentrations up to 1.6 M after Mg2+ gradient elution of immunoaffinity-precipitated exosome preparations. The Dis3 (Rrp44) subunit, instead, is eluted at approximately 0.6 M Mg2+ (Allmang, et al., 1999). Even though Dis3 (Rrp44) is more salt- sensitive than the other core subbunits, it is still seen as a fundamental subunit because it is present in both cytoplasmic and nuclear complexes and is crucial for cell viability (Mitchell et al., 1997; Allmang et al., 1999b). Native mass – spectrometry analysis of the yeast exosome has revealed that Dis3 (Rrp44) associates to the core ring structure on the opposite side of the trimeric cap of RNA binding proteins (Hernandez et al., 2006).

The exosome in the nucleus

In the nucleus the 3´- 5´ turnover of RNA by the exosome is a important issue regarding the large mass of intron sequences generated from splicing, the products derived from cleavage and trimming events, and the erroneous RNA and other RNA species (reviewed by Butler, 2002). Besides the turnover function, the exosome is the key component of mRNA surveillance in the nucleus. The nuclear exosome has the capacity to distinguish between substrates that are destined for RNA maturation, where only a few nucleotides are supposed to be removed in a controlled and precise manner, and RNAs that are destined to be retained in the nucleus and rapidly degraded (Galy et al., 2004; Hilleren et al., 2001). Overall, the functions of the exosome can be divided in two groups: degradative and processive. When it comes to the processive functions of RNA species in the nucleus, the exosome is involved in the synthesis of many RNAs such as: snRNA, snoRNA, rRNA and tRNA (reviewed by Butler, 2002; Raijmakers et al., 2004). The first discovered function of the yeast exosome was the maturation of the 5.8S ribosomal RNA (rRNA) (Mitchell et al., 1996; 1997). In both yeast and human the 35S pre-rRNA is cleaved and trimmed into mature 18S, 5.8S and 25S rRNAs. The 5´A0 ETS generated from the cleavage of the 35S pre-rRNA are completely degraded by the exosome and Rrp6. Moreover, Rrp6 and the exosome have been reported to be important players in converting 7S and 5.8s+30 into the mature 5.8S s. The exosome and Rrp6 carry out the exonucleolytic removal of the 3´end of the 7S up until the last 30 nucleotides, which then are trimmed away by Rrp6 (reviewed by Butler, 2002). The nuclear exosome is also an important factor in the 3´end processing of snRNA and snoRNA. This mechanism indirectly affects mRNA splicing and rRNA biogenesis as many snRNA (like U1, U4 and U5) are components of the spliceosome, and snoRNAs are involved in pre-rRNA processing (Allmang et al., 1999a; van Hoof et al., 2000; Kufel et al., 2000; Mitchell et al. 2003). The degrading function of the nuclear exosome concerns nuclear mRNA surveillance, which is connected to the poly (A) tail of different RNA species. In E. coli, it is well established that poly (A) addition enhances degradation by

27 the 3´-5´exonucleases PNPase and RNase II as the poly (A) tail serves as a landing platform for these exonucleases. This function of the poly (A) tail had been considered to be exclusive of bacteria. In eukaryotes, the poly (A) tail is known to stimulate export to the cytoplasm, has a stabilizing function, and promotes translation. In the yeast mRNA surveillance mechanism, the correct poly (A) tail is a mark of a successfully processed transcript and it prevents degradation by the exosome (Burkard and Butler, 2000; Hilleren et al., 2001; Milligan et al., 2005). However, the view that the poly (A) tail acts merely as a stabilizer has been substantially modified during the last few years. Three reports published during the year 2005 showed that snRNAs, snoRNAs, some rRNAs and tRNAs are polyadenylated prior to degradation by the exosome (LaCava et al., 2005; Vanacova et al., 2005; Wyers et al., 2005). This was shown when depleting the exosome component Rrp6 in a S. cerevisiae mutant strain (Wyers et al., 2005), which resulted in increased levels of polyadenylated RNAs. This study also detected an enrichment of RNA Pol II transcripts with poly (A) tails derived from intergenic regions that had never been seen before. These cryptic unstable transcripts (CUTs) were believed to be degraded continuously by the exosome, and therefore had been undetected before. Wyers et al. (2005) could also conclude that it was not the ordinary Pap-1 polyadenylation machinery that was responsible for the polyadenylation of CUTs. The CUTs were stabilized in Δtrf4 and Δair1/Δair2 mutant yeast strains, and this lead to the conclusion that the novel Trf/Air/Mtr4 (TRAMP) complex was responsible for their polyadenylation (LaCava, et al., 2005; Vanacova, et al., 2005; Wyers et al., 2005). TRAMP has strong polyadenylation activity and addition of TRAMP to an exosome preparation enhanced its degradative activity. It has been proposed that TRAMP is not involved in exosome- mediated processing, but works as a co-activator during nuclear RNA decay by the exosome (LaCava et al., 2005; Vanacova et al., 2005; Wyers et al., 2005). In addition, the exosome has been shown to degrade a class of substrates that are synthesized by RNA pol II and generated both sense and antisense upstream of promoters of annotated transcribed sequences: the promoter upstream transcripts (PROMPTs) (Preker et al., 2008).

28

Figure 3. General overview of the exosome functions in

cytoplasm and nucleus.

The exosome in the cytoplasm

The single substrate for the cytoplasmic form of the exosome is mRNA. The exosome performs 3´end turnover of normal mRNAs (van Hoof et al., 2000; Anderson and Parker, 1998). It is also involved in the surveillance and thereby degradation of mRNA in the cytoplasm, as it is part of the NMD pathway. Besides this, the exosome is involved in the no-go decay pathway (NGD), where it degrades the cleaved 5´fragment generated from stalling of the translating ribosome (the 3´fragment is degraded by the 5´exonuclease Xrn1). The stalling of the translation can be a consequence of mRNA damage or erroneous ribosome function (Doma and Parker 2006; Tollervey 2006). It has also been shown in Drosophila melanogaster that the exosome degrades the 5´fragments that are produced after RNA interference (RNAi) cleavage (Orban and Izaurralde 2005). The cytoplasmic exosome also degrades mRNAs with specific A+U rich sequence elements (AREs) in human cells. The mRNAs that have ARE sequences are very unstable and their abundance can vary rapidly. ARE-containing mRNAs include growth factors, proto-oncogene products, cytokines and lymphokines (reviewed by van Hoof & Parker, 2002). These mRNA are degraded both in the 5´-3´and 3´-5´manner upon decapping and poly (A) tail shortening, respectively (Muhlrad et al., 1994). The antiviral protein ZAP is able to recruit cytoplasmic exosome and thereby mediate degradion of viral mRNA that contains ZAP-binding elements (ZREs) (Guo et

29 al., 2007) Furthermore, it has been suggested that the exosome is also involved in the regulated turnover of oligouridylated Histone mRNAs during the late S- phase in mammalian cells (Mullen and Marzluff, 2008)

Activation of the exosome

In contrast to the archeal exosome, yeast exosome has fairly low exonuclease activity in vivo (Mitchell et al., 1997). The archeal exosome readily degrades single-stranded RNAs that come in contact with the S1 pore. A large number of cofactors that can activate the yeast exosome have been reported. The different cofactors that activate the exosome and process the target RNAs make it possible for the exosome to distinguish between substrates. The cofactors are thought to be important for targeting the substrates to the exosome, and for unwinding the substrate RNA. Moreover, the need for cofactors may also serve as protection against redundant RNA degradation. The activating cofactors can be divided in two major classes: those that bind specific sequences in the RNA target, and those that identify specific structures of the target RNA-protein complex. The sequence-specific Nrd1 (nuclear binding protein) has been shown to activate RNA degradation by the exosome both in vivo and in vitro in yeast (Arigo et al., 2006; Vasijeva and Buratowski, 2006). Several different RNA species such as snRNA and snoRNAs contain Nrd1-binding sites in their precursor forms (Steinmetz et al., 2001; Arigo et al., 2006). It has been proposed that if the Nrd1 binding site in the immature RNAs is not removed by ordinary processing, the RNA will be completely degraded by the exosome. This mechanism constitutes a quality control of the processing of several types of RNA substrates, including mRNAs, snRNAs and snoRNAs (reviewed by Houseley et al., 2006). The ARE- mediated degradation in human cells is another example of sequence-specific degradation. This pathway activates the exosome in two ways. Either the exosome directly associates with the ARE-binding proteins, for example AUF1/hnRNP D or TTP (Chen et al., 2001; Gherzi et al., 2004), or via the RNA helicase RhAU that associates with the exosome (Tran et al., 2004). The non-specific cofactors give a more complex picture because the structure or sequence of the substrates for these factors lacks obvious resemblances. The RNA substrate has to be single stranded to be able to pass the central channel, and here the RNA helicases play an important role in unwinding the substrate for translocation through the S1 pore (de la Cruz et al., 1998). In yeast, the DExH-box RNA helicase Mtr4 was the first cofactor of this type to be found. The first report of the role of Mtr4 concluded that Mtr4 is required for normal rRNA processing via its interaction with the exosome (de la Cruz et al., 1998). Besides the function in trimming and degradation in rRNA biogenesis, Mtr4 is also part of the Trf-Air-Mtr4 (TRAMP) polyadenylation complex (LaCava et al., 2005; Vanacova et al., 2005; Wyers et al., 2005).

30 As mentioned above, the TRAMP complex polyadenylates different species of RNA by adding short poly (A) tails at the 3' end. The addition of the short poly (A) tail recruits and activates the exosome for degradation. Two forms of the complex have been found in yeast, TRAMP4 and TRAMP5, depending on whether the complexes contain Trf4 or Trf5. Trf4 and Trf5 are distributive poly (A) polymerases that belong to the same polymerase family as Pap1p (Wyers et al., 2005; la Cava et al., 2005; Vanacova et al., 2005). The two zinc-knuckle proteins Air1 and Air2 are also part of the TRAMP and are suggested to provide the RNA-binding activity that Trf4 and Trf5 lack (Vanacova et al., 2005; Wyers et al., 2005). The TRAMP pathway can be summarized as follows. Air1 or Air2 bind to the substrate via their RNA- binding domains, and thereafter Mtr4 recruits and activates the exosome (Wyers et al., 2005; la Cava et al., 2005; Vanacova et al., 2005). TRAMP affects the exosome in two ways. On one hand, the addition of a poly (A) tail by the TRAMP marks the RNA as a better substrate for the exosome, presumably the A-tail serving as a landing platform for the exosome. On the other hand, TRAMP increases the enzyme activity of the exosome (reviewed by Houseley and Tollervey, 2006). Another non-specific co-activator is the putative RNA-binding protein Rrp47. In the nucleus, Rrp47 delivers snoRNA and rRNA to Rrp6 as part of the maturation process (Mitchell et al., 2003b). There are links between the pathways that involve sequence-specific and non- specific co-activators. For instance, Nrd1 can be co-precipitated with Trf4 and Air2, and also with exosome subunits (Vasiljeva and Buratowski, 2006). In addition, U14 snoRNAs with extended 3´ends accumulate in strains that are defective in Nrd1, TRAMP4 or exosome components (la Cava et al., 2005; Arigo et al., 2006; Vasiljeva and Buratowski, 2006). These observations show that the different types of pathways are not independent. However, the links between them are not known yet. The cytoplasmic cofactors are common for all exosome substrates. The cytoplasmic cofactors are the Ski complex and Ski7. The Ski complex comprises the putative DExH-box RNA helicase Ski2 (homologue to Mtr4), Ski3 and Ski8 (Brown et al., 2000; Araki et al., 2001). Besides being important for the cytoplasmic mRNA turnover pathway, the Ski complex and Ski7 are involved in both NMD and non-stop decay pathways together with the exosome (Takahashi et al., 2003; Mitchell and Tollervey, 2003; van Hoof et al., 2002).

The role of the nuclear exosome in mRNA surveillance

The exosome is co-purified with the elongation factors Spt6 and Spt5 as well as with RNA Pol II in Drosophila (Andrulis et al., 2002). In situ studies have also shown that exosome subunits co-localize with Spt6 at several loci with active transcription. Furthermore, chromatin immunoprecipitation experiments on heat shock genes (hsp70 and hsp26) revealed that exosome subunits are recruited to both of these genes. The amount of exosome subunits and Spt6

31 associated with gene sequences (e.g. heat-shock genes) increased upon gene activation, and it has been argued that this might be to monitor the quality of the growing transcript during elongation (Andrulis et al., 2002). According to this hypothesis, the exosome would degrade erroneous and/or abortive RNAs with a free 3´end that did not manage to elongate efficiently, which would prevent clogging of the mRNA synthesis and processing machineries (Andrulis et al., 2002). Additionally, the exosome core subunit Rrp4 has been shown to associate with the nascent-pre-mRNP cotranscriptionally (see Paper II in this Thesis). A specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. This was shown in the insect model systems Drosophila melanogaster and Chironomus tentans. It implies that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. The interaction with the nascent pre-mRNP is suggested to serve as part of cotranscriptional surveillance (Hessle et al., 2009). It is known that mRNAs with splicing defects are down regulated by the surveillance machinery. mRNAs expressed from mutated splicing reporter constructs are degraded in wild type cells (Burgess and Guthrie, 1993). Yeast splicing mutants have decreased mRNA levels and notably without an increase of the corresponding unspliced transcript. Pre-mRNAs that fail to efficiently assemble the spliceosome do get exported to the cytoplasm and are degraded in the cytoplasm presumably by the cytoplasmic mRNA turnover machinery. On the other hand, pre-mRNAs that are defective in the splicing reaction after spliceosome assembly are retained in the nucleus (Legrain and Rosbash, 1989; Rain and Legrain, 1997; Rutz and Seraphin, 2000). By inactivating the exosome in the yeast prp2-1 splicing mutant strain, there is a 50-fold increase in levels of pre-mRNA in the nucleus. When Rat1 was inactivated in the same prp2-1 mutant, the mRNA levels become stabilized to a much lesser extent. This data suggested that the exosome serves as a tool for degrading the pre- mRNA with aberrant splicing that is retained in the nucleus (Bousquet- Antonelli, 2000). In Drosophila, the exosome subunit Rrp6 is required for the release from the transcription site of transcripts that are defective in splicing, as part of cotranscriptional splicing surveillance. The same study showed that the presence of splicing mutations in the mRNA down-regulates the transcription of the mutated gene (Paper III, Eberle et al., 2010). Not all transcripts are spliced co-transcriptionally, and the nuclear retention factors Mlp1 and Mlp2 have been reported to retain unspliced mRNA in the nucleus (Galy et al., 2004). Mlp1 and Mlp2 are in contact with the nucleoporin Nup60 and provide a surveillance checkpoint at the nuclear pore. The exosome subunit Rrp6 might serve also in this surveillance pathway, as Rrp6 and Mpl1 have been reported to interact physically. The model proposed is that intron- containing mRNAs associate with Mlp1 at the nuclear rim, where they can be degraded by the exosome (reviewed by Dimaano and Ullman, 2004). The polyadenylation process is also a target for mRNA surveillance by Rrp6 and the exosome. The yeast Rrp6 physically interacts with the poly (A) polymerase Pap1p and the nucleo-cytoplasmic shuttling protein Npl3p (Bukard and Butler, 2000; Lee, et. al, 1996). Supporting the importance of Rrp6 and the

32 exosome in the surveillance of the polyadenylation, it has been reported that mRNAs with either hypo- or hyper-adenylated 3´ends are retained close to the transcription site in yeast (Hilleren et al., 2001). In rrp6Δ and rrp4-1 mutant strains, the retention is partially disrupted and export is restored, suggesting that the exosome is part of this quality control (Hilleren et al., 2001). Furthermore, the exosome has a role in the surveillance of defective polyadenylation; not only the final structure of the transcript but also the rate of processing can be sensed by the exosome (Milligan et al., 2005). When a defective pap1-5 mutant protein is expressed, both cleavage and polyadenylation can still occur to a certain extent, but polyadenylation is slow and pre-mRNA surveillance is triggered. According to the model presented by Milligan et al. (2005), low polyadenylation rates are sensed through a mechanism that requires Rrp6 and the RNA helicase Mtr4, and the transcripts are degraded by the exosome (Milligan et al., 2005). Recently, a connection between nuclear RNA surveillance, the exosome, and RNA pol II transcriptional termination was reported in mammalian cells. The work was done in a mouse murine erythroleukemia (MEL) cell line expressing β-globin constructs either wild type or defective in processing. When the unprocessed β-globin transcripts were retained at the site of transcription, these defective pre-mRNAs remained associated to RNA pol II at the DNA template. Moreover, this association was Rrp6 dependent (de Almeida et al., 2010). The surveillance machinery also senses defects in mRNP assembly. As described in the export and retention part of this thesis, yeast THO/sub2 mutants accumulate nascent transcripts of the HSP104 gene in the nucleus (Libri et al., 2002). In the THO/sub2 mutants, the 3´end formation and the release of the transcript from transcription sites were impaired, leaving the defective mRNAs retained at or close to the transcription sites (as part of the mRNA surveillance system) and subsequently degraded by the nuclear exosome (Rougemaille et al., 2007).

Present investigation

Aim of this thesis

The main objective of this thesis has been to study the RNA exosome and its role in nuclear mRNA surveillance. By the time we started this project, very little was known regarding the functions of the metazoan exosome. We realized that we had the opportunity to contribute to the detailed knowledge by studying the exosome with Chironomus tentans and Drosophila melanogaster as model systems.

33

Model systems

The diptera Chironomus tentans is a non-biting midge that has successfully been used as model system to study gene expression in situ. The life cycle of this organism includes four stages: egg, larvae, pupa and adult animal. At 18°C the life cycle takes approximately six weeks. The larval stage is divided in four instars, each lasting between five and ten days. By the fourth instar, the larvae are about 10-15 mm long and the salivary glands consist of 35-40 large cells coating a central lumen.

Polytene chromosomes and nuclear organization Many insect tissues develop polyteny. This implies that the cells undergo repeated rounds of replication without separating the sister chromatids, forming large polytene chromosomes, and that the cells grow instead of dividing. As a result, the nucleus of a Figure 4. Illustration of adult and polytene cell is considerably larger larvae life stages of Chironomus. than a typical diploid nucleus, and Miall and Hammond, 1900. Printed exhibits giant polytene chromosomes with permission from Oxford instead of chromosome territories as University Press. mammalian nuclei do. The salivary gland cells of Chironomus are a typical example of polytene cells. In these cells, the interchromatin space is unusually spacious and devoid of chromatin. Polytene nuclei have typical nucleoli but other sub nuclear compartments have not been described in Chironomus. The polytene chromosomes were first described in Chironomus by EG Balbiani in 1881. The extraordinarily large nucleus of the salivary gland cells in C. tentans contains four chromosomes, each consisting of 8000-16000 chromatides aligned (reviewed by Hägele, 1975; Case and Daneholt 1977). Each chromosome has a characteristic banding pattern created by the level of compaction of the chromatin. At the sites of active transcription, the chromatin is expanded; sometimes forming lateral expansions or puffs, and the alternation of expanded and compact chromatin regions gives rise to the distinct banding pattern (Beermann, 1952).

34 BR genes The most actively transcribed protein-coding genes in the salivary glands of C. tentans are the Balbiani ring (BR) genes. The mRNP particles generated from BR genes are transported to the cytoplasm where they are translated into vast polypeptides (about 1 MDa) that are secreted to give rise to the proteinaceous tubes where the larvae reside in (reviewed by Wieslander, et. al., 1994; Daneholt, 2001). There are five BR genes in Chironomus. Four of them -BR1, BR2.1, BR2.2 and BR6- are exceptional in size (approximately 40 kb). The BR2 genes have four short introns: three are located in close proximity to the 5´end and the fourth close to the 3´end, with the approximately 35 kb long exon IV in between. The pre-mRNA of BR3 holds 38 introns, and this precursor transcript gives rise to an mRNA of 5.5kb (reviewed by Daneholt, 2001). The BR pre-mRNAs go through typical pre-mRNA processing. They are capped, spliced, cleaved and polyadenylated, and are exported to the cytoplasm as mature mRNPs (Visa, et al., 1996; Bauren, et al., 1998; Bauren and Wieslander, 1994). As transcription proceeds, the nascent BR pre-mRNAs appear as growing RNP fibers along the chromatin loop. In electron microscopy, the transcribed BR genes can be seen as chromatin loops decorated by RNP fibers of increasing length along the gene. As transcription progresses and the RNP fiber grow, the end of the fiber is folded into a globular structure that increases in size along the loop. By the end of the gene, the globular part of the pre-mRNP is approximately 50 nm in diameter (Daneholt, et al., 1982). The size and abundance of the BR1 and BR2 mRNPs makes it possible to identify them and to visualize the location, the assembly and the export of the BR mRNP particles from the gene through the nuclear pore complex into the cytoplasm in situ. This has been shown in numerous studies using electron microscopy (reviewed by Mehlin and Daneholt, 1993; Daneholt, 2001). The BR system also provides for the possibility to map the distribution of proteins along the transcribed gene. For instance, the mapping of splicing factors along the transcribing BR genes was done using an anti-snRNP antibody and immuno-electron microscopy (Kiseleva, et al., 1994). Another example is the mapping of the histone acetyltransferase p2D10 (Sjölinder et al., 2005). Besides using electron microscopy, it is also possible to localize factors of interest using the immuno-fluorescence technique on whole salivary glands (for instance Sjölinder, et al., 2005).

Drosophila melanogaster Chironomus tentans as is a powerful system for immunohistochemical studies, mainly at the electron microscopic level, because of the possibility of visualizing the expression of the BR gene. The work described in this thesis has engaged Chironomus as the main model system. However, it has limitations when genomic sequences are needed, since the Chironomus genome is not fully sequenced. Also, the Chironomus system lacks tools for genetic manipulation. To date, no Chironomus cell lines have been established that are possible to transfect (e.g. in order to express epitope- tagged proteins or do RNA interference). This is why we turned to the well-

35 established Drosophila model system for RNA interference experiments and ChIP assays. The Drosophila S2 cell line, derived from embryos, is a useful tool for heterologous protein expression. In some of the studies presented in this thesis, S2 cells were stably transfected with plasmid encoding V5-tagged dRrp4 and dRrp6 under the control of an inducible promoter. Furthermore, the salivary glands of Drosophila larvae, which also contain polytene chromosomes, were used in some cases for comparative purposes.

Summary of the projects included in this thesis

Paper I: The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation.

We have established a chromatin immuno precipitation (ChIP) protocol for C. tentans salivary glands. In order to do this, we chose to work with antibodies against three proteins that associate with the transcribed BR genes through different types of molecular interactions. First, we studied the chromatin- remodeling factor Brm. Second, were also analyzed the histone H3 trimethylated at lysine 4 (meH3). This protein is expected to be in direct contact with the promoter proximal sequences (Santos-Rosa et al., 2002; Kouskouti and Talianidis, 2005). The third protein, Rrp4, is a component of the core RNA exosome. We first showed that Brm, Rrp4 and meH3 associate with the transcribed BR genes in the salivary gland polytene chromosome of C. tentans by analyzing preparations of isolated chromosomes by immunofluorescence. The ChIP method had previously been applied to salivary glands of D. melanogaster (Legube et al., 2006). However, the available protocols did not provide satisfactory results in C. tentans and therefore we introduced some modifications. The major parameters that we adjusted were the time of sonication and the concentration of detergents. We report the conditions suitable for ChIP with each of the three antibodies. Anti-Rrp4 and anti-meH3 worked under highly stringent conditions, while anti-Brm required a sodium deoxycholate (DOC) concentration below 0.5% (1% for the other two antibodies). We found sonication conditions that are suitable to extract chromatin from C. tentans salivary glands, and we could conclude that the optimal conditions for the ChIP experiment have to be individually calibrated for the proteins studied. Brm has been proposed to be involved in nucleosomes remodeling at promoter regions (Soutoglu and Talianidis, 2002) and in pre-mRNA splicing (Batsché et al., 2006). We carried out immuno-EM experiments combined with ChIP on salivary glands in order to study the distribution of Brm along the BR1 gene in the salivary glands of C. tentans. Combined results from the immuno-

36 EM and ChIP revealed that Brm is associated with proximal, middle and distal parts of BR genes and not restricted to the promoter region. In summary, in Paper I we have established optimal conditions for ChIP on chromatin extracted from the salivary glands of C. tentans. This is interesting because it allows us to analyze by ChIP the same genes that we can analyze by immuno-EM, which provides a valuable way of confirming data. This method has been applied in the studies described in Papers II and IV.

Paper II: The exosome associates cotranscriptionally with the nascent pre-mRNP through interactions with heterogeneous nuclear ribonucleoproteins.

In D. melanogaster it has been shown that the exosome is linked to RNA polymerase II by physically interacting with the elongation factors Spt5/6. This interaction provides for cotranscriptional recruitment of the exosome to transcribed genes. This interaction is suggested to be a part of cotranscriptional pre-mRNA surveillance (Andrulis et al., 2002). In this study we wanted to scrutinize the mechanisms of recruitment of the nuclear exosome to protein-coding genes. We focused on the core exosome subunit Rrp4 for studies in C. tentans and D. melanogaster. The sequences of the exosome proteins of C. tentans were not known. To determine the sequence for Rrp4 we cloned the cDNA for C. tentans Rrp4. The full-length cDNA for Ct-Rrp4 contains an ORF of 291 amino acids that encodes a protein of 32.8 kDa. BLAST comparisons showed that Ct-Rrp4 was 61 and 63% identical to the A. gambiae and D. melanogaster orthologues respectively. An anti- antibody was raised in rabbit. Immunofluorescence experiments on chromosomes isolated from C. tentans salivary glands showed that Ct-Rrp4 is associated with transcribed genes and intensely stain the nucleolus. DRB treatment showed that the association of Ct- Rrp4 with the chromosomes is transcription dependent. RNAse A treatment revealed that the association of Ct-Rrp4 with transcribed loci was to a large extent dependent on RNA. However, a fraction of Rrp4 associated to the chromosomes was insensitive to the RNAse A treatment, suggesting two modes of interaction of Rrp4 with the chromosomes. Immuno-EM experiments showed that Ct-Rrp4 is present along the entire transcription unit of BR genes and this association was confirmed with ChIP on salivary gland chromatin. The precise location of Ct-Rrp4 in the active BR genes was analyzed by immuno- EM. We could show that a significant fraction of Ct-Rrp4 was associated with the nascent pre-mRNPs without being in contact with the transcription machinery. The concentration of Rrp4 along the gene was not constant. The level of Ct- Rrp4 was significantly higher in the proximal part of the BR gene than the distal. This led us to analyze the distribution of the exosome along other genes with different gene structural features. For this we carried out ChIP experiments in order to detect proteins attached to DNA as well as proteins that interact with the nascent transcript. For the ChIP experiments we constructed a

37 Drosophila S2 cell line that expressed a V5-tagged version of Rrp4 under an inducible promoter. We used an anti-V5 tag antibody and quantification of the immunoprecipitated DNA was based on real-time PCR assays using primes sets along five different genes. Ct-Rrp4 was present in all regions of all genes analyzed. We could conclude that Ct-Rrp4 distribution is not directly correlated with the intron-exon structure of the genes or with the levels of RNA Pol II. We could also show a similar, although not identical (see Paper IV), distribution of Rrp6 along the genes. This observation suggests that the results with Ct-Rrp4 reflected to quite an extent the behavior of the catalytic exosome. The RNase treatments, immuno-EM and ChIP results suggested an additional mode of interaction of the exosome to transcribed genes different from the physical interaction with the transcription machinery reported by Andrulis and colleagues (Andrulis et al. 2002). We preformed coimmunoprecipitation assay in order to determine whether Rrp4 interacts with proteins that are associated with the nascent pre-mRNA. The coimmunoprecipitated proteins were identified by high-throughput mass spectrometry. Parallel experiments were also carried out to identify interaction partners for two other exosome subunits: Rrp6 and Ski6. In all three cases, the hnRNP protein Hrp59 was found significantly enriched in the exosome-bound fraction. With cell fractionation and immunoprecipitation methods we could confirm that Hrp59 and Rrp4 interact with each other and we showed that this interaction takes place on the chromosomes. We investigated the functional significance of the Rrp4-Hrp59 interaction by silencing the expression of Hrp59 by RNA interference (RNAi) in S2 cells that express Rrp4-V5. We analyzed the association of Rrp4 with transcribed genes by ChIP after the Hrp59 silencing. The results from the RNAi experiments showed that the depletion of Hrp59 interferes with the association of Rrp4 with the transcribed genes. This suggests that Hrp59 mediates the interaction between the exosome and the pre-mRNP. Hrp59 is an abundant protein that binds to transcripts from many different genes (Kiesler et al. 2005). By binding to Hrp59, the exosome is tethered to nascent transcripts cotranscriptionally.

Paper III: Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing- defective genes.

The surveillance system in eukaryotes prevents the expression of aberrant transcripts. An early checkpoint of the surveillance machinery acts at the transcription site and prevents the release of transcripts with processing defects. The exosome subunit Rrp6 is required for the retention in but whether Rrp6 is needed for mRNA surveillance in higher eukaryotes was not known. The human β-globin gene had been used in previous studies to analyze the surveillance mechanisms that operate during transcription. Wild type (wt) and mutant (mut) versions of the β-globin gene were used to study the retention of

38 unprocessed pre-mRNAs at the transcription site in mammalian cells (Custodio et al., 1999; 2007). We have applied this approach in D. melanogaster in order to study the nuclear retention of unspliced transcripts. Plasmids carrying the wt and mut β-globin genes under the control of an inducible metallothionein promoter were stably transected into Drosophila S2 cells. Fluorescent in situ hybridization (FISH) experiments were preformed in order to determine whether Drosophila has a mechanism for retention of unspliced mRNAs at the site of transcription. In this type of experiments, transcription is inhibited with Actinomycin D, and the release of transcripts from the transcription sites is quantified. The rationale of the approach is that defects in mRNA biogenesis prevent the release of the newly synthesized RNAs, which accumulate at the gene and can then be detected by FISH. Our results implied that indeed, there was a retention mechanism in Drosophila. To test whether Rrp6 was needed for the nuclear retention, as it is in S. cerevisiae, we repeated the FISH analysis after silencing the expression of Rrp6 by RNAi in S2 cells expressing wt and mut β-globin genes. In the cells expressing wt β-globin, the depletion of Rrp6 did not have any significant effect on the retention of β-globin transcripts, whereas in the cells expressing mut β-globin the retention was less efficient after depletion of Rrp6. From these results we concluded that Rrp6 is involved in the nuclear retention of the unspliced mut β-globin RNAs. We also analyzed the processing of the 3’ end of the transcripts in order to better understand the consequences of having splice-site mutations. A series of experiments showed that the mut β-globin mRNA were cleaved and polyadenylated, and that the levels of polyadenylation in wt and mut β-globin transcripts were similar. We found that in the S2 cell line that expresses the mut β-globin gene, the levels of mut β-globin pre-mRNA and mRNA were remarkably lower than the corresponding wt transcripts To investigate whether the difference in pre- mRNA/mRNA levels was due to impaired transcription, we preformed ChIP with an antibody against the CTD in order to quantify the density of pol II in mut and wt β-globin genes. The ChIP experiments showed that the mut β- globin gene was less efficiently transcribed than the wt β-globin gene, suggesting that splicing influences transcription. The reduced CTD density was observed close to the promoter and in downstream regions of the gene, which suggests that transcription is affected at the level of initiation. Additional ChIP experiments with antibodies against two post translational histone modification (H3K4me3 and H3Ac) marking actively transcribed chromatin resulted in the proposal that the impaired transcription of the mut β-globin gene is accompanied by changes in chromatin structure involving specific histone modifications. In summary, the results presented in Paper III show that the co-transcriptional surveillance in insects includes retention mechanisms similar to the ones described previously in mammalian cells. We also showed that Rrp6 participates in the retention mechanism. A recent report has shown that this is also the case in mammalian cells (de Almeida et al. 2010). Finally, the study in Paper III suggests that nuclear surveillance does not only act by degrading

39 defective transcripts, but also by down-regulating their synthesis. Indeed, the presence of splicing defects in the mRNA feeds back on the transcription machinery and reduces the transcription initiation rates.

Paper IV (manuscript): The intranuclear localization of the exoribonuclease Rrp6 in insect cells.

In this study we wanted to get further insight into the functions of the exosome subunit Rrp6 in metazoans. For this, we used the model systems of C. tentans and D. melanogaster. The spotlight for this study was the nuclear distribution of Rrp6 in the salivary gland cell nucleus of C. tentans, and how Rrp6 is associated to the BR genes. We cloned a partial cDNA for Ct-Rrp6, we expressed the partial recombinant protein in E. coli, and we used the recombinant protein to raise polyclonal antibodies in rabbit. We analyzed the nuclear localization of Rrp6 in salivary gland cells. We showed that the anti-Rrp6 antibody stains the nucleoplasm intensely compared to the cytoplasm. We showed that Rrp6 localizes to actively transcribed genes and to numerous loci in the polytene chromosomes. Additionally, and interestingly, Rrp6 was enriched in discrete nuclear foci that we refer to as RNBs for Rrp6-rich Nuclear Bodies. The anti-Rrp6 antibody was also used in immuno-EM (IEM) studies in order to analyze the localization of Rrp6 in the active BR genes. The IEM experiments revealed Rrp6 binding to the nascent BR particles and suggested that Rrp6 is transported to the nuclear pores as part of the mRNP. We then analyzed the association of Rrp6 with genes in D. melanogaster in order to determine if the Rrp6 distribution along genes resembled the distribution pattern of Rrp4 as previously reported (Paper II). The experiments were preformed using S2 cells that expressed V5-tagged version of the Drosophila Rrp6 (as described above in Paper II). ChIP experiments were preformed and analyzed. The distribution pattern of Rrp6 was similar to that of Rrp4 in the sense that both proteins were detected in all the regions of all the analyzed genes, from promoter to polyadenylation sites. However, the pattern of Rrp6 distribution did not fully follow the pattern of Rrp4 (Hessle et al., 2009), which suggests that a fraction of Rrp6 that locates at transcription units is not associated to the core exosome. Next, we wanted to investigate the enrichment of Rrp6 in discrete nuclear bodies in the salivary gland cells of C. tentans. A series of immunofluorescence experiments were preformed to clarify the nature of these bodies. We could determine that the bodies were not enriched with nascent RNA and were not dependent on active transcription. Moreover, the RNBs did not contain Rrp4, suggesting that the function of the bodies is independent from the exosome. To further elucidate the nature of the RNBs, we did a series of immunofluorescence experiments with double labeling. Results from double labeling with typical marker proteins for Cajal bodies and nuclear speckles

40 made us exclude the possibility that the RNBs were any of these two nuclear bodies. Double labeling experiments also showed that Rrp6 and the small ubiquitin-like modifier (SUMO) colocalized in RNBs. Furthermore the mRNA export factor Rae1 with roles in mitotic spindle assembly (Blower et al., 2005) was also colocalized with Rrp6 in RNBs. Additionally, SUMO and Rae1 were colocalized in RNBs. These results imply that Rrp6 is enriched in SUMO in C. tentans and that these bodies constitute a novel nuclear structure in the cell nuclei of this organism. We also wanted to verify the presence of RNBs in D. melanogaster. As described earlier in this thesis, D. melanogaster exhibits Cajal bodies as well as histone locus bodies (HLBs). Again, we did a series of immunofluorescence experiments with double staining. We could show that the RNBs exist in D. melanogaster separately from Cajal bodies and HLBs. We could also show that SUMO is enriched in nuclear bodies. However, double-staining experiments with anti-Rrp6 and anti-SUMO revealed that RNBs and SUMO NBs are different entities, as Rrp6 and SUMO rarely colocalize in D. melanogaster.

Conclusions

Paper I:

• We have established optimal conditions for Chromatin Immuno Precipitation on chromatin extracted from the salivary glands of Chironomus tentans.

• With ChIP and immuno-EM we have mapped the distribution of the SWI/SNF chromatin-remodeling factor Brahma (Brm) along the entire BR 1 gene and we have found that Brm is located along the entire BR transcription unit, which suggests that Brm has additional roles apart from regulating transcription initiation.

• We have also detected Rrp4 in association with the BR1 gene.

Paper II:

• A significant fraction of the core exosome subunit Rrp4 is associated with nascent pre-mRNPs in C. tentans.

• The mRNA binding protein Hrp59 interacts in vivo with the core exosome subunits Rrp4, Rrp6 and Ski6 in Drosophila.

• Depletion of Hrp59 by RNAi in Drosophila reduces the levels of Rrp4 at transcription sites.

41 • Considering the results included in this paper, we have presented a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNPs.

Paper III:

• We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster cells.

• Insect cells have mechanisms that prevent the release of splicing- defective transcripts from the gene.

• Rrp6 is needed for the retention of splicing-defective transcripts at the gene.

• Splicing-defective RNAs are cleaved and polyadenylated but the transcription of the genes that code for splicing-defective RNAs is down regulated.

• At least two surveillance responses that operate cotranscriptionally in insect cells. One requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other one acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

Paper IV:

• Immuno-EM and immunofluorescence experiments revealed the nuclear distribution of Rrp6 in the salivary glands of C. tentans. We show that Rrp6 is associated with mRNPs during transcription and during intranuclear transport.

• Rrp6 is enriched in novel discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster.

• In C. tentans the Rrp6-rich nuclear bodies (RNBs) colocalize with SUMO, whereas in D. melanogaster we have identified RNBs and SUMO-rich bodies as different entities.

• RNBs do not contain the core exosome subunit Rrp4, which suggests that their function is not related to the functions of the exosome.

• RNBs also contain Rae1, an evolutionarily conserved protein involved in mRNA export and in regulation of cell cycle progression.

42 Acknowledgements

This study was carried out at the Department of Molecular Biology and Functional Genomics at Stockholm University. I would like to thank every person within our department and other departments that have in one or another way contributed to this thesis.

First of all, I would like to thank my supervisor Neus Visa. Your ability to contaminate me with your enthusiasm is amazing. Countless times I have walked in to your office with heavy shoulders and after a few minutes come out of there full of energy. Your way of looking at a project/problem/result from every angle is very impressive. I also want to thank you for always being patient and respectful when my personal life has come in the way for lab work.

Two persons deserve special treatment here: Johan Ohlson and Sara Jupiter. I want to thank the two of you for being the best friends one can have. I miss all the fun we had during coffee breaks, lunches and parties. Lucky me that Sara married my brother and Johan also moved söder om söder.

Members of Neus lab group, past and present. Especially new-Johan Waldholm for also having an Iphone4 and, of course, for being a good and incredibly fun friend! Also, thanks to lab members/co-authors/friends Andrea, Emilia, Anne, and Rebecca.

All past and present colleagues at molbio. Especially the gorgeous MÖ-girls: Chammiran “Tabita” Daniel, Helene Wahlstedt and Ylva Ekdahl. Thanks Kerstin Bernholm, for sharing all the tricks about lab work. Petra Björk, for going the extra mile to ask how things are. Anne Beskow-the human dynamo. Pat Young, for all laughs and being the clear-eyes, peanut-butter-m´n´ms and fossile dealer. Britta, for taking care of the larvae and for making lab work easier. Marie, Pär, Lars, Åsa, Micke, Kristian, Shiva, Maria, Solveig, Tomas, Ylva Engström, Widad, Clara, Gunnel, Josefine, Margaretha, Britt-Marie, Gilad, Uli, Zhi, Nevin, Rula, Jamie, Fredrik, Christina, Marina, and Marc. If I left anyone out, I will take the kitchen-week for you!

My beloved family (in alphabetical order to avoid a family-feud): Albin, Beata, Curt, David, Gisela, Johanna, Johannes, Kalle, Klisse, Lotta, Maja, Mamma, Martina, Mia, Pappa, Sara (igen…), Siri, Sixten, Sven and Ulla. I am incredibly fortunate to have the all of you.

Anna, Björn och Ulric, ni tre är det bästa i mitt liv. Jag älskar er!

43

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