Regulation of pre-mRNA splicing and mRNA degradation in

Yang Zhou

Department of Molecular This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7601-749-4 Cover photo by Yang Zhou Electronic version available at: http://umu.diva-portal.org/ Printed by: Print & Media Umeå Umeå, Sweden 2017

by 千利休

Every single encounter never repeats in a time. -Sen no Rikyu

Table of Contents

ABSTRACT ...... ii APPENDED PAPERS ...... iii INTRODUCTION ...... 1 Pre-mRNA splicing ...... 1 Splicing and ...... 1 The pre-mRNA Retention and splicing complex ...... 6 Nuclear export of mRNAs...... 7 ...... 7 Translation initiation ...... 9 General mRNA degradation ...... 11 mRNA quality control mechanisms ...... 12 Nonsense-mediated mRNA decay ...... 14 NMD substrates and premature translation termination ...... 14 NMD mechanisms ...... 17 NMD inactivation and nonsense suppression ...... 19 RESULTS AND DISCUSSION ...... 21 CONCLUSIONS ...... 30 ACKNOWLEDGEMENT ...... 31 REFERENCES ...... 33 Paper I-III ...... 55

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ABSTRACT

Messenger are transcribed and co-transcriptionally processed in the nucleus, and transported to the . In the cytoplasm, mRNAs serve as the template for synthesis and are eventually degraded. The removal of sequences from a precursor mRNA is termed splicing and is carried out by the dynamic . In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated. The pre-mRNA retention and splicing (RES) complex is a spliceosome- associated that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N4-acetylcytidine (ac4C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 , which is required for the formation of ac4C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs. The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in , and its expression is controlled by the NMD pathway via a uORF in the 5’ . We show that the reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg2+ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg2+ exporter Mnr2p and the mitochondrial Mg2+ exporter Mme1p for degradation. In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg2+ concentration by controlling the expression of Mg2+ transporters.

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APPENDED PAPERS

This thesis is based on the following papers, which are referred to by their Roman numerals (I-III). Paper I is reproduced with permission from the publisher.

I. Zhou Y, Chen C, Johansson MJ. 2013. The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression. Nucleic Acids Res. 41:5669-5678.

II. Zhou Y, Johansson MJ. 2017. The pre-mRNA retention and splicing complex controls expression of the Mediator subunit Med20. RNA Biol. doi: 10.1080/15476286.2017.1294310.

III. Zhou Y, Johansson MJ. The nonsense-mediated mRNA decay pathway controls the expression of magnesium transporters in Saccharomyces cerevisiae. (Manuscript)

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INTRODUCTION

Life of a messenger RNA

In 1961, messenger RNA (mRNA) was identified with evidence from a phage-infected bacterial system as an unstable intermediate that carries genetic information from to and was the missing link between DNA and protein (Brenner et al. 1961). Following the discovery of mRNA, the concept of triplet codons was demonstrated, and the various codons were deciphered (Crick et al. 1961; Leder and Nirenberg 1964). These early findings elucidated the nature of and were later shown to be universally applicable.

Protein-coding genes in are transcribed by RNA polymerase II (Pol II) and generate precursor mRNAs (pre-mRNAs). Several pre-mRNA processing events are executed co-transcriptionally, including 5’ end capping, 3’ end formation and splicing (Bentley 2005). Capping occurs shortly after initiation (Jove and Manley 1984; Rasmussen and Lis 1993). Briefly, capping enzymes are recruited to the C-terminal of the Pol II large subunit, where the tri-phosphate at the 5’ end of a nascent transcript is converted to a di-phosphate group. A mono- phosphate (GMP) molecule is transferred to the 5’ di-phosphate end, and a methyl group is introduced at the N7 position of the guanosine base (Shatkin and Manley 2000; Zhai and Xiang 2014). The formation of the mRNA 3’ end in eukaryotes consists of two steps. A free hydroxyl end is generated by an endonucleolytic cleavage near the 3’ end of a nascent transcript, and poly(A) polymerase initiates the synthesis of a poly(A) tract at the cleavage site (Bentley 2005). The length of the poly(A) varies among different species. In Saccharomyces cerevisiae, the poly(A) tail is ~70-90 (Eckmann et al. 2011; Parker 2012). Both the m7GpppN cap and the poly(A) tail structures have an important impact on translation and transcript stability (Eckmann et al. 2011; Topisirovic et al. 2011).

Pre-mRNA splicing

Splicing and introns In eukaryotes, a precursor mRNA (pre-mRNA) may contain intervening sequences (introns) that are usually not translated. Pre-mRNA splicing is a nuclear process that involves intron removal and the ligation of coding

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sequences () (Padgett et al. 1986). In metazoans, a given pre-mRNA usually has several introns and it can be alternatively spliced to generate mRNA isoforms with different stabilities and coding potentials (Nilsen and Graveley 2010). An event may involve exclusion, intron retention or the use of alternative splice sites, and it can alter the structure and function of the final protein product (Latorre and Harries 2017). Disruption of splicing or alternative splicing contributes to several human diseases (Ward and Cooper 2010; Latorre and Harries 2017).

Unlike most eukaryotes, S. cerevisiae has only ~282 intron-containing open reading frames (ORFs), and most of them have a single intron that is primarily located adjacent to the 5’ end of the (Ares lab yeast intron database: http://intron.ucsc.edu/yeast4.3/) (Spingola et al. 1999), so budding yeast is a simple model organism to study the regulation of splicing. A large fraction of intron-containing transcripts in yeast are pre-mRNAs from highly expressed ribosomal protein genes, suggesting that splicing frequently occurs in yeast (Ares et al. 1999; Lopez and Seraphin 1999). In general, an intron sequence carries three conserved cis-acting elements – the 5’ splice site, the 3’ splice site, and the branchpoint (Will and Luhrmann 2011). In higher eukaryotes, the intron usually has a between the branchpoint and the 3’ splice site (Will and Luhrmann 2011). The most common sequences for the 5’ splice site, the branchpoint, and the 3’ splice site in yeast are GUAUGU, UACUAAC, and U/CAG respectively (Figure 1A) (Spingola et al. 1999). Alterations of these sequence elements affect the splicing efficiency (Jacquier and Rosbash 1986; Seraphin et al. 1988; Kandels-Lewis and Seraphin 1993; Luukkonen and Seraphin 1997).

Although it is tempting to assume that spliceosomal introns have always been -specific and that more and more introns were inserted into eukaryotic genomes during the time course of eukaryotic evolution, the origin as well as the gain or loss of introns are still open to debate (Roy and Gilbert 2006). The introns-early hypothesis suggests that the common ancestors of and eukaryotes contained introns, which were then lost from prokaryotic genomes (Penny et al. 2009). Conversely, the introns- late model postulates that intron sequences were inserted into originally intron-less eukaryotic genes after the divergence of prokaryotes and eukaryotes (Logsdon 1998). There are also two general opinions regarding the differences in intron number among different eukaryotic organisms. Some believe that the common ancestors of eukaryotes had only few introns and the vast majority of introns were gained during eukaryotic evolution

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(Logsdon et al. 1998; Roy and Gilbert 2006). Others argue that the common ancestors of eukaryotes were intron-rich, and the current intron-poor species such as S. cerevisiae have experienced massive intron loss during evolution (Roy and Gilbert 2006).

Figure 1. Pre-mRNA splicing by the U2 snRNP-dependent spliceosome (A) Schematic representation of conserved sequence elements in yeast introns. The consensus sequences at the 5’ splice site, branchpoint and 3’ splice site are indicated. (B) Schematic representation of the U2-dependent spliceosome assembly. The spliceosomal complexes at each stage of the assembly are indicated. The stepwise interaction of the is shown. The exons are shown as boxes and the intron is shown as line or lariat. The two catalytic steps of splicing are indicated.

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The chemical mechanism of pre-mRNA splicing is two sequential trans- esterification reactions, which are catalysed by a multi-megadalton ribonucleoprotein (RNP) complex known as the spliceosome (Jurica and Moore 2003). The spliceosome machinery is highly dynamic, which means that its conformation and composition undergo several changes during a single splicing event (Figure 1B). The assembly of the spliceosome requires five -rich, small nuclear RNP (U snRNP) particles (U1, U2, U4, U5 and U6 snRNPs) and numerous non-snRNP protein factors (Sperling et al. 2008; Will and Luhrmann 2011). In addition to the major U2 snRNP- dependent spliceosome, a U12 snRNP-dependent also exists in metazoans (Patel and Steitz 2003), and these will not be discussed in this thesis. Each of the snRNP particles consists of a snRNA, seven Sm (termed after Smith antigen) or like-Sm (LSm) , and a set of U snRNP-specific protein factors (Migliorini et al. 2005; Stark and Luhrmann 2006; Will and Luhrmann 2011). The canonical assembly of the spliceosome starts with the binding of the substrate pre-mRNA by the U1 snRNP by base- pairing between the 5’ end of the U1 snRNA and the 5’ splice site of the intron. The non-snRNP factors SF1 and U2 auxiliary factor (U2AF) interact with the branchpoint sequence and the 3’ end of the intron sequence, respectively, forming the spliceosomal E complex (Jurica and Moore 2003; Stark and Luhrmann 2006; Wahl et al. 2009; Will and Luhrmann 2011). The U2 snRNP replaces SF1 and associates with the branchpoint by base- pairing between the U2 snRNA and the branchpoint sequence, resulting in the formation of the spliceosomal A complex (Will et al. 2001). Notably, the base-pairing between the U2 snRNA and the branchpoint is stabilized by the highly conserved U2 snRNP protein complex SF3a and SF3b (Gozani et al. 1996). U4/U6 snRNP is formed by extensive base-pairing between the U4 and U6 snRNAs, and forms U4/U6.U5 tri-snRNP with U5 snRNP (Sander et al. 2006). The U4/U6.U5 tri-snRNP is recruited to the spliceosomal A complex, resulting in the formation of the spliceosomal B complex (Wahl et al. 2009). At the B complex stage, U5 snRNA contacts exons at both the 5’ and the 3’ ends of the intron, and the 3’ end of the U6 snRNA base-pairs with the 5’ end of the U2 snRNA (Wahl et al. 2009). The U6 and U2 snRNAs undergo major rearrangements during the activation stage of the B complex. As a result, the 5’ end of the U6 snRNA base-pairs with the 5’ splice site of the intron and displaces the U1 snRNA. U4/U6 snRNA base-pairing is also interrupted and replaced by extensive base-pairing between the U6 and U2 snRNAs (Madhani and Guthrie 1992; Sun and Manley 1995; Sashital et al. 2004). After these conformational changes, the entire U1 snRNP as well as the U4 snRNA and a large portion of the U4/U6.U5 tri-snRNP protein factors

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are released from the spliceosome, giving rise to the activated spliceosomal Bact complex (Fabrizio et al. 2009; Will and Luhrmann 2011). The Bact complex is further activated by ATPase Prp2p, generating the B* complex (Kim and Lin 1996). The B* complex catalyses the first chemical reaction of splicing, which is an attack by a 2’-hydroxyl group of an at the branchpoint on the phosphodiester bond at the 5’ splice site (Sperling et al. 2008). This reaction generates a free 5’ exon and a lariat intron-3’ exon intermediate (Padgett et al. 1986). The spliceosome then undergoes compositional and conformational changes and generates the spliceosomal C complex, which catalyses the second splicing reaction (Fabrizio et al. 2009). The 3’-hydroxyl group of the 5’ exon attacks the phosphodiester bond at the 3’ splice site, resulting in the ligation of exons and a free lariat intron (Padgett et al. 1986). Notably, the second reaction requires ATPase Prp16p and Prp22p, which promote the rearrangement from the B complex to the C complex (Schwer and Guthrie 1992; Schwer and Gross 1998). After the second reaction, the spliceosome dissociates and the mRNA is released (which also requires Prp22p) (Schwer and Gross 1998). The lariat intron is released, debranched and rapidly degraded, while the snRNPs as well as other spliceosomal components are recycled for another round of splicing (Wahl et al. 2009; Hesselberth 2013). In yeast, inefficiently spliced pre- mRNAs are subject to degradation by nuclear endonuclease Rnt1p, nuclear exonuclease Rat1p, nuclear , exonuclease Xrn1p, and the nonsense-mediated mRNA decay pathway (Bousquet-Antonelli et al. 2000; Danin-Kreiselman et al. 2003; Egecioglu et al. 2012; Sayani and Chanfreau 2012).

The regulation of splicing relies on both cis-acting elements and trans- acting factors. In addition to the conserved 5’ splice site, branchpoint and 3’ splice site, there are other cis-regulatory elements that function as splicing enhancers or splicing silencers (Smith and Valcarcel 2000; Wang and Burge 2008). These sequence elements exist in both introns and exons, and associate with stimulating or suppressing protein factors during constitutive splicing and alternative splicing (Wang and Burge 2008). Adenosine-to- inosine editing in either an exon or an intron also affects the splicing efficiency in metazoans and has been suggested as a regulatory mechanism for splicing and gene expression (Rueter et al. 1999; Higuchi et al. 2000). A variety of protein factors are required for the regulated splicing of specific transcripts during the cycle and the development of different organisms (Shin and Manley 2004). Examples of splicing-regulatory proteins include the Sex-lethal protein that functions during sex determination in Drosophila

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melanogaster and Mer1p that is required for meiosis-specific splicing in budding yeast (Valcarcel et al. 1993; Munding et al. 2010).

The pre-mRNA retention and splicing complex In addition to the snRNPs, numerous non-snRNP protein complexes are engaged at different stages of spliceosomal assembly. The pre-mRNA REtention and Splicing (RES) complex represents one of the non-snRNP complexes. The RES complex is a heterotrimeric complex that consists of Bud13p, Snu17p (also known as Ist3p), and Pml1p (Dziembowski et al. 2004). Originally identified in yeast, the RES complex is also present in humans and it associates with spliceosome prior to the first catalytic step of splicing (Dziembowski et al. 2004; Deckert et al. 2006; Bessonov et al. 2008). In yeast, the RES complex is suggested to associate with U2 snRNP and interact with the intron (Gottschalk et al. 2001; Dziembowski et al. 2004; Wysoczanski et al. 2014). Snu17p serves as the core subunit and binds both Bud13p and Pml1p independently (Gottschalk et al. 2001; Dziembowski et al. 2004; Wang et al. 2005b; Brooks et al. 2009). Previous studies suggest that Bud13p and Snu17p are engaged in splicing, while Pml1p promotes nuclear retention of the unspliced pre-mRNAs (Dziembowski et al. 2004; Tuo et al. 2012).

The yeast RES complex is not essential for splicing in the sense that yeast cells lacking any of the RES factors are viable (Dziembowski et al. 2004). However, deletions of the RES genes do give rise to several different phenotypes. Yeast cells lacking any RES factors show growth defects that are exacerbated at elevated temperatures (Dziembowski et al. 2004). Interestingly, the growth defects observed in bud13Δ and snu17Δ cells are more severe than the growth defect in pml1Δ cells, suggesting that the growth phenotypes of individual RES-mutants are correlated with their respective importance to splicing (Dziembowski et al. 2004). Recently, the cause of the temperature sensitive (Ts) growth phenotype of bud13Δ and snu17Δ cells has been identified as the inefficient splicing of the MED20 pre- mRNA, a gene coding for a subunit of the Mediator complex (see Paper II). Diploid cells in which both copies of the BUD13 or SNU17 gene are deleted show a haploid-like budding pattern, and a MATα-like mating pattern (Ni and Snyder 2001; Schmidlin et al. 2008). These phenotypes are caused by the inefficient splicing of the MATa1 pre-mRNA, a gene codes for a repressor of haploid-specific genes (Schmidlin et al. 2008; Tuo et al. 2012). Notably, the RES complex is also required for the Mer1p-activated splicing of AMA1 and

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MER2 pre-mRNAs during meiosis (Spingola et al. 2004; Scherrer and Spingola 2006).

Although genome-wide studies of RES-mutants suggest that the complex is necessary for the efficient splicing of several transcripts, both genome- wide studies and studies of individual RES targets failed to identify a common feature for RES-dependent transcripts (Clark et al. 2002; Khanna et al. 2009). It is still debatable whether the RES complex or RES factors recognize certain sequences within introns, but it is not a generally required protein complex for pre-mRNA splicing in yeast.

Nuclear export of mRNAs Mature mRNAs are exported to the cytoplasm via nuclear pore complexes (NPCs). The nuclear export of mRNAs is mediated by Mex67p-Mtr2p, a hetero-dimer that interacts with both mRNA and NPC (Reed and Hurt 2002). Aberrantly processed mRNAs are usually subject to nuclear mRNA degradation or retention. Nuclear retention of unspliced pre-mRNAs is facilitated by spliceosome-associated factors, as well as several NPC- connected proteins (e.g., Mlp1p, Mlp2p, Pml39p) (Schmid and Jensen 2008). Some inefficiently spliced pre-mRNAs manage to escape to the cytoplasm— a phenomenon termed leakage.

Translation Protein translation is regarded as the last step of the central dogma, in which the information carried by mRNAs is decoded and utilized to assemble amino acids into polypeptides. In eukaryotes, the basic mechanisms of protein synthesis are well conserved, and studies in yeast have provided critical insights into the fundamental process and regulation of translation (Altmann and Linder 2010; Dever et al. 2016). It is generally accepted that protein synthesis occurs in the cytoplasmic compartment of eukaryotic cells. The possibility of nuclear translation has been hypothesized (Iborra et al. 2001; Apcher et al. 2013; Anton and Yewdell 2014), but the supporting data for nuclear translation was shown to be insufficient (Nathanson et al. 2003; Dahlberg and Lund 2004; Kuperwasser et al. 2004). Translation is usually divided into four stages: initiation, elongation, termination, and ribosome recycling (Kapp and Lorsch 2004). In this thesis, an overview of initiation will be briefly described.

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Figure 2. Pathway of eukaryotic translation initiation The schematic representation is based on the cap-dependent scanning model. The mRNA, initiator tRNA, 40S and 60S subunits, and eukaryotic initiation factors are shown as different shapes. The stepwise interaction of the mRNA, initiation factors and ribosomal subunits is shown. The canonical initiation pathway begins with the formation of 43S pre-initiation complex. The 43S complex then attaches to the 5’ end of the mRNA (facilitated by the eIF4F complex) and scans the mRNA for an optimal AUG codon. Upon AUG recognition, the initiation complex undergoes compositional and conformational changes, followed by joining of the 60S subunit

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and the production of the 80S ribosomal complex. The GTP-hydrolyses by eIF2 and eIF5B and the release of eIFs are indicated. The eIF1is also shown with dashed lines because it is unclear if eIF1 is released from the initiation complex upon AUG recognition. See the text for more details.

Translation initiation Translation initiation is the process that generates 80S ribosomes that synthesize proteins. Translation initiation of most eukaryotic mRNAs follows the cap-dependent ‘scanning’ mechanism (Figure 2), which begins with the formation of the 43S pre-initiation complex (PIC) (Kozak 1978; Jackson et al. 2010; Hinnebusch 2011; Hinnebusch et al. 2016). The formation of the 43 PIC involves eukaryotic initiation factor 1 (eIF1), eIF1A, the eIF3 complex, the ternary complex and likely eIF5 (Jackson et al. 2010; Hinnebusch 2011). The ternary complex (TC) consists of the GTP-bound eIF2 complex and the Met initiator tRNA (Met- tRNA i ). During ribosome recycling, the eIF3 complex, eIF1 and eIF1A are recruited to the 40S ribosomal subunit, where they (including eIF5) stimulate the assembly of the 43S PIC (Jackson et al. 2010; Hinnebusch 2011; Dever et al. 2016). The TC subsequently attaches to the 40S subunit and the eIFs, forming the 43S PIC. The next step of initiation is the binding of the eIF4F complex to the 5’ cap of the mRNA, which prepares the transcript for the attachment of the 43S PIC to the 5’ end of the mRNA. The eIF4F complex comprises the RNA eIF4A, the m7G cap binding protein eIF4E, the scaffold protein eIF4G and the poly(A) binding protein (Pab1p in yeast) (Jackson et al. 2010; Hinnebusch 2011). The helicase eIF4A unwinds secondary structures in the 5’ cap-proximal region of mRNA. The helicase activity of eIF4A is ATP-dependent and stimulated by eIF4G and eIF4B or eIF4H (Jackson et al. 2010; Hinnebusch 2011). The subsequent ribosomal attachment to mRNA in mammals is facilitated by the interaction between eIF4G and eIF3, while this step is likely promoted by eIF4G-eIF5 interaction in yeast (LeFebvre et al. 2006; Dever et al. 2016). Notably, the interaction between eIF4G and the poly(A) binding protein causes the mRNA to form a ‘closed-loop’ structure, which is important for the contact of eIF4F with the 5’ end of the mRNA (Kahvejian et al. 2005; Jackson et al. 2010; Park et al. 2011). After it is loaded on the mRNA, the 43S complex starts scanning the 5’ untranslated region (UTR) of the mRNA, until it reaches an AUG start codon. The scanning is promoted by eIF1, eIF1A and eIF3. The helicase eIF4A together with eIF4G and eIF4B unwind secondary structures in the 5’-UTR, allowing the 43S PIC to thread along the mRNA (Jackson et al. 2010). In addition to eIF4A, other ATP-dependent also contribute during scanning, including DHX29 in mammals and Ded1p in yeast (Iost et al. 1999; Pisareva et al. 2008). The 43S PIC is

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usually arrested at the first AUG codon in an optimal context, which is often referred to as the ‘first AUG rule’ (Kozak 1978; Hinnebusch 2011). In vertebrates, the for efficient initiation is GCC(A/G)CCAUGG, but a strong preference exists for A at position -3 (in relative to the AUG codon) (Kozak 1987a; Yun et al. 1996). In yeast, the 5’- UTR sequences are AU-rich, and the sequences surrounding the start codon also strongly prefer an A at position -3 (Hinnebusch 2011). Alteration of the at position -3 decreases translation in both mammals and yeast, suggesting that this residue is functionally conserved in eukaryotes (Hinnebusch 2011). The ribosome switches from an ‘open’ scanning conformation to a ‘closed’ conformation (i.e., locked on the mRNA) when the Met anticodon of Met- tRNA i matches an AUG start codon (Maag et al. 2005). During this process, the affinity of eIF1 for the 43S complex is decreased (it may still associate with the 43S subunit), and the GTP in TC is hydrolysed (Unbehaun et al. 2004; Maag et al. 2005). GTP hydrolysis is achieved via GTPase activity in the eIF2 γ-subunit, which is induced by the GTPase activating protein eIF5 (Hinnebusch 2011). The AUG recognition leads to the formation of the 48S PIC, which is followed by the joining of the 60S ribosomal subunit. GTP-bound eIF5B is recruited to the 48S complex where it promotes the joining of the 60S subunit and the release of eIF1A, eIF3, eIF2-GDP and possibly eIF1 (Pestova et al. 2000; Unbehaun et al. 2004). During this process, eIF1A stimulates GTP hydrolysis by eIF5B, which in turn promotes the release of eIF5B from the ribosome (Jackson et al. 2010; Dever et al. 2016). After the joining of the 60S subunit and the release of eIFs, an elongation-competent 80S ribosome is completed.

When a 5’-proximal AUG is surrounded by unfavourable context, it will be bypassed by the scanning ribosome. This exception to the first AUG rule is termed ‘leaky scanning’ (Kozak 1986), and it allows the ribosome to initiate translation at a downstream AUG codon. Reinitiation is another mechanism that involves initiation at a downstream AUG. The reinitiation mechanism allows the 40S subunit to resume scanning after translation has been terminated at an upstream ORF (uORF) and reinitiate at a downstream AUG codon (Kozak 1987b). Reinitiation is inefficient if the uORF is close to the downstream ORF, and the efficiency improves as the distance between the uORF termination codon and the downstream AUG codon increases (Kozak 1987b; Abastado et al. 1991). This length dependence of reinitiation suggests that the reassembly of the 43S complex requires sufficient time and TC before it reaches the downstream AUG (Kozak 2005; Hinnebusch 2011). These two mechanisms are frequently utilized in uORF-regulated

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translation in eukaryotes (Gaba et al. 2001). One well-studied example of reinitiation is the of the transcriptional activator Gcn4p in yeast (Hinnebusch 2005). The GCN4 5’-UTR has 4 uORFs, and uORF1 and uORF4 are essential for its regulated expression. Briefly, the ribosome translates uORF1 and reinitiates at uORF2, 3 or 4, but dissociates from the GCN4 mRNA before initiation at the main ORF under non- starvation conditions. Under amino acid-starvation conditions, the ribosome fails to reinitiate at uORF2 to 4 after translating uORF1 and scans past them due to a decrease in the TC concentration. The ribosome subsequently rebinds to TC before reaching the main ORF, and reinitiates at the GCN4 AUG (Hinnebusch 2005).

Although rarely, the eukaryotic ribosome can also initiate translation at near-cognate non-AUG codons (Peabody 1989; Chang and Wang 2004; Tang et al. 2004). The initiation at a non-AUG codon is usually weak and strongly dependent on an optimal sequence context (Kozak 2005; Chen et al. 2008a).

So far, I briefly described some aspects of the cap-dependent scanning mechanism for translation initiation in eukaryotes. A cap-independent internal initiation mechanism also exists, and it is often referred to as internal ribosome entry site (IRES)-mediated translation initiation. The early studies on IRES-mediated initiation were focused on the translation of a subset of viral mRNAs (Jang et al. 1988; Pelletier et al. 1988). It has also been suggested that cellular mRNAs in eukaryotes may utilize the same mechanism for translation initiation under certain conditions (Reineke and Merrick 2009; Jackson 2013). However, an alternative explanation and alternative mechanisms may exist for the observed cap-independent initiation (Gilbert 2010).

General mRNA degradation The mRNA that has fulfilled its duty for protein synthesis is usually subject to turnover (Moore 2005). The general cytoplasmic turnover of eukaryotic mRNAs is initiated by the shortening of the poly(A) tail at the 3’ end, a process termed deadenylation (Decker and Parker 1993). In yeast, deadenylation is catalysed by two protein complexes, the CCR4-NOT complex and the PAN2-PAN3 complex (Brown and Sachs 1998; Tucker et al. 2001). The CCR2-NOT complex consists of 9 subunits. Two of them, Ccr4p and Caf1p show exonuclease activity, and Ccr4p is the main catalytic subunit (Daugeron et al. 2001; Tucker et al. 2002; Goldstrohm et al. 2007).

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The PAN2-PAN3 complex exists as a heterotrimer comprising a Pan2p molecule and a Pan3p dimer, with Pan2p as the catalytic subunit and Pan3p as the regulatory subunit (Boeck et al. 1996; Brown and Sachs 1998). It has been suggested that PAN2-PAN3 is responsible for the initial trimming of the poly(A) tail, which is a rapid shortening from ~90 residues to ~65 residues, while CCR4-NOT is responsible for the further deadenylation (Parker 2012), which is consistent with the observation that the poly(A) binding protein Pab1p promotes the activity of PAN2-PAN3 but inhibits CCR4-NOT (Boeck et al. 1996; Tucker et al. 2002). After deadenylation, transcripts in yeast cells are subjected to exosome-mediated 3’ to 5’ degradation or decapping followed by exoribonuclease Xrn1p-mediated 5’ to 3’ degradation (Hsu and Stevens 1993; Muhlrad et al. 1994; Mitchell et al. 1997; Anderson and Parker 1998). The degradation mechanism involving decapping has been suggested as the major pathway of mRNA degradation in yeast cells (Muhlrad et al. 1995). Briefly, a Dcp1p-Dcp2p dimer cleaves the m7GpppN cap, creating a m7GDP molecule and a mRNA with a 5’ monophosphate, which is then degraded by Xrn1p (Parker 2012). Dcp2p is the catalytic subunit and its decapping activity can be promoted by Dcp1p (Dunckley and Parker 1999; She et al. 2008). Alternatively, mRNAs can be degraded by the cytoplasmic exosome in a 3’ to 5’ direction after deadenylation. The yeast cytoplasmic exosome is a multi-subunit protein complex, and its catalytic subunit Rrp44p has both endonuclease and exonuclease activities (Dziembowski et al. 2007; Lebreton et al. 2008; Schaeffer et al. 2009). Degradation of the mRNAs by the exosome also requires the Ski complex, which consists of Ski2p, Ski3p and Ski8p (Anderson and Parker 1998; Brown et al. 2000; Wang et al. 2005a). The Ski complex interacts with the exosome via the bridging protein Ski7p (van Hoof et al. 2000; Araki et al. 2001; Wang et al. 2005a). After the degradation of the mRNA body by the exosome, the leftover 5’ cap (m7GpppN) is hydrolysed by the scavenger decapping enzyme Dcs1p (Liu et al. 2004). mRNA quality control mechanisms In addition to mechanisms for general mRNA turnover, eukaryotic cells have evolved several quality control pathways that ensure the accuracy of gene expression by eliminating incorrectly processed or non-functional transcripts (Ghosh and Jacobson 2010). These mRNA surveillance mechanisms not only prevent the accumulation of aberrant transcripts and the generation of potentially toxic proteins but are also involved in the post-transcription regulation of wild-type mRNAs (Kervestin and Jacobson 2012). In this thesis, I will describe three cytoplasmic quality control pathways.

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The no-go decay (NGD) pathway is a cytoplasmic quality control mechanism that targets translationally active mRNAs on which ribosomes stall during translation (Doma and Parker 2006; Harigaya and Parker 2010; Parker 2012; Siwaszek et al. 2014). Degradation by the NGD pathway was initially observed in yeast mRNA harbouring an artificial stem-loop (Doma and Parker 2006). The function of NGD involves the two protein factors Dom34p and Hbs1p, which have been suggested as homologues of the eukaryotic release factors eRF1 (Sup45p in yeast) and eRF3 (Sup35 in yeast) respectively (Carr-Schmid et al. 2002; Doma and Parker 2006; Graille et al. 2008). Briefly, the stalled ribosome is recognized by Dom34p, which binds to the empty ribosomal A site (Becker et al. 2011). Dom34p together with Hbs1p promotes the release of the peptidyl-tRNA and the dissociation of the ribosome subunits, which is a process of similar to the translation termination mediated by release factors (Doma and Parker 2006; Chen et al. 2010; Shoemaker et al. 2010; van den Elzen et al. 2010). Endonucleolytic cleavage of the transcript is carried out by a yet unknown nuclease (Doma and Parker 2006; Parker 2012). The resulting 5’ fragment is degraded by the cytoplasmic exosome, while the 3’ fragment is degraded by Xrn1p (Doma and Parker 2006). The nascent is then degraded via the ubiquitin- proteasome pathway (Graille and Seraphin 2012; Parker 2012).

Another cytoplasmic quality control mechanism termed non-stop decay (NSD) also exists, and it targets transcripts that lack an appropriate translation termination codon (Frischmeyer et al. 2002; Vasudevan et al. 2002; Klauer and van Hoof 2012). The substrates of the NSD pathway usually arise when transcription aborts or when occurs within an ORF, which is often referred to as premature polyadenylation (Frischmeyer et al. 2002; Vasudevan et al. 2002; Cui and Denis 2003; Klauer and van Hoof 2012). Premature polyadenylation occurs in approximately 1% of tested random yeast cDNA clones (Graber et al. 1999; Frischmeyer et al. 2002). The function of the NSD pathway involves the cytoplasmic exosome, as well as the Ski complex (Ski2p, Ski3p and Ski8p) and Ski7p (van Hoof et al. 2002). Briefly, a translating ribosome arrives at the 3’ end of a non-stop mRNA and stalls, which is likely detected by Ski7p, leading to the recruitment of the Ski complex and the exosome, and finally to the 3’ to 5’ degradation of the non-stop transcript (van Hoof et al. 2002; Klauer and van Hoof 2012; Parker 2012). The protein product generated from a non-stop transcript is degraded by the ubiquitin-proteasome pathway (Graille and Seraphin 2012; Klauer and van Hoof 2012; Parker 2012).

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Nonsense-mediated mRNA decay The nonsense-mediated mRNA decay (NMD) pathway is a cytoplasmic surveillance mechanism that targets mRNAs that contain premature termination codons (PTCs) for rapid degradation (Leeds et al. 1991; Peltz et al. 1993). Originally identified in S. cerevisiae, the NMD pathway is also found in higher eukaryotes and is thought to be evolutionarily conserved from yeast to humans (Pulak and Anderson 1993; Lykke-Andersen et al. 2000; Gatfield et al. 2003; Schweingruber et al. 2013; He and Jacobson 2015). In fact, the NMD pathway has been shown to exist in all eukaryotes that have been investigated (Chen et al. 2008b; Delhi et al. 2011; Schweingruber et al. 2013). The NMD pathway is often referred to as a post- transcriptional regulatory mechanism for gene expression, since it is involved in many biological processes, including cell proliferation and growth (Weischenfeldt et al. 2008; Avery et al. 2011; Lou et al. 2014), embryonic development and differentiation (Medghalchi et al. 2001; Gong et al. 2009; McIlwain et al. 2010; Bruno et al. 2011), stress response (Gardner 2010; Sakaki et al. 2012), innate immunity (Gloggnitzer et al. 2014), and neuronal activity (Giorgi et al. 2007; Colak et al. 2013; He and Jacobson 2015). The NMD pathway shows an increasing clinical relevance. in genes that encode NMD factors lead to neural disorders and intellectual disability (Tarpey et al. 2007; Nguyen et al. 2012; Jolly et al. 2013; Nguyen et al. 2013; Xu et al. 2013). It has also been estimated that ~30% of human inherited genetic diseases, as well as many types of cancer, are attributable to the presence of PTCs, which are generated from nonsense mutations or frame-shifts (Frischmeyer and Dietz 1999; Mendell and Dietz 2001). In some PTC-mediated diseases, the inhibition of NMD function can be beneficial and therapeutic, as it promotes the translation of nonsense transcripts and the generation of partially or even fully functional protein products (Linde et al. 2007; Gonzalez-Hilarion et al. 2012; Peltz et al. 2013; Keeling et al. 2014).

NMD substrates and premature translation termination The NMD pathway targets a wide range of eukaryotic transcripts. Studies in different organisms have revealed that approximately 3% to 10% of the mRNAs from a typical eukaryotic show accumulation upon NMD inactivation (Lelivelt and Culbertson 1999; He et al. 2003; Mendell et al. 2004; Rehwinkel et al. 2005; Guan et al. 2006; Metzstein and Krasnow 2006; Wittmann et al. 2006; Weischenfeldt et al. 2008; Ramani et al. 2009; Wittkopp et al. 2009; Yepiskoposyan et al. 2011; Tani et al. 2012; Weischenfeldt et al. 2012). The substrates of the NMD pathway fall into

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several categories, including transcripts from nonsense alleles (Leeds et al. 1991), inefficiently spliced pre-mRNAs that enter the cytoplasm (He et al. 1993), some mRNAs that contain uORFs (Johansson and Jacobson 2010), mRNAs that undergo frameshifts (Morris and Lundblad 1997; Belew et al. 2011), mRNAs that undergo PTC-generating alternative splicing (Ni et al. 2007; Weischenfeldt et al. 2012), mRNAs derived from rearranged genetic loci (Li and Wilkinson 1998; Wang et al. 2002), mRNAs with atypically long 3’-UTRs, bicistronic mRNAs (He et al. 2003), transcripts derived from (He et al. 2003), transcripts derived from transposable elements (He et al. 2003), and long noncoding RNAs (Kurihara et al. 2009). The NMD pathway not only eliminates non-functional and harmful transcripts but also regulates the stability of wild-type transcripts (Conti and Izaurralde 2005; Kervestin and Jacobson 2012; He and Jacobson 2015). Accordingly, the broad range of NMD substrates suggests that the NMD pathway has a significant biological impact.

Although NMD-targeted transcripts can be classified into several categories, they share a common feature in that translation is prematurely terminated. Both normal and premature translation termination events involve the recognition of a at the ribosomal A site, while NMD is triggered only when the translational machinery recognizes a stop codon as premature. Therefore, it is important to address the features that distinguish premature translation termination from normal translation termination (Figure 3). In eukaryotes, translation termination at a stop codon (UAA, UAG or UGA) requires the coordinated function of two classes of release factors (Alkalaeva et al. 2006). Class I release factor eRF1 (Sup45p in yeast) functions as a tRNA, and recognizes a stop codon and binds to the ribosomal A site, resulting in hydrolysis of the peptidyl-tRNA at the ribosomal P site (Kisselev and Buckingham 2000). Class II release factor eRF3 (Sup35p in yeast) is a GTPase that stimulates the hydrolytic activity of eRF1 (Kisselev and Buckingham 2000). The factor eRF1 hydrolyses peptidyl-tRNA in the absence of eRF3 but slowly (Eyler et al. 2013). The release factors eRF1 and eRF3 interact with each other and function as a complex (Kisselev and Buckingham 2000). The GTPase activity of eRF3 is intrinsically repressed, and it requires the association of both eRF1 and the ribosome to stimulate GTP hydrolysis (Frolova et al. 1996). GTP hydrolysis by eRF3 is followed by a conformational change of eRF1 (Alkalaeva et al. 2006). The conformational change moves the Gly-Gly-Gln motif of eRF1 towards the peptidyl transferase centre of the ribosome, where it induces the hydrolysis of the peptidyl-tRNA (Alkalaeva et al. 2006). After the peptide is released,

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the recycling factor ATP-binding cassette subfamily E member 1 (Rli1p in yeast) interacts with eRF1, and triggers the dissociation and recycling of the ribosomal subunits (Pisarev et al. 2010; Becker et al. 2012). Notably, an interaction between eRF3 and the poly(A)-binding protein (Pab1p in yeast) has been shown to promote normal translation termination (Hoshino et al. 1999; Cosson et al. 2002). Overexpression of Pab1p enhances translation termination efficiency, while a lack of it reduces termination efficiency in human cells (Cosson et al. 2002; Ivanov et al. 2008).

Figure 3. Mechanistic differences between normal translation termination and premature termination in yeast (A) In normal termination, the release factors eRF1 and eRF3 are recruited to the terminating ribosome and induce peptide release. The interaction between Pab1p and eRF3 promotes efficient termination. Dissociation and recycling of the terminating complex is mediated by Rli1p in yeast. (B) In premature termination, the recruitment of release factors is inefficient, due to the lack of proper stimulation from the 3’-UTR. Upf1p is recruited (or activated) by premature termination. Upf2p and Upf3p are subsequently recruited to the terminating ribosome and regulate the activity of Upf1p. The Upf proteins facilitate the recruitment of Dcp1p-Dcp2p decapping enzyme and the dissociation of the premature termination complex.

Premature termination is often referred to as aberrant termination, since it is considered slower and less efficient than normal termination (Figure 3B). Previous studies showed that ribosomes can be detected at PTCs by toe- print assays, but not at normal termination codons, suggesting that the

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ribosome pauses at a PTC (Amrani et al. 2004; Peixeiro et al. 2012). PTCs are also more vulnerable to readthrough by near-cognate tRNAs compared to normal termination codons (Welch et al. 2007; Peltz et al. 2013). However, it is yet unclear at which step premature termination is delayed. The inefficiency of premature termination is likely due to a lack of necessary components in the mRNP complex, for example, poly(A)-binding protein (Schweingruber et al. 2013; He and Jacobson 2015).

NMD mechanisms

NMD factors

In yeast, the NMD pathway requires the three evolutionarily conserved protein factors Upf1p, Upf2p and Upf3p (also respectively known as Smg2p, Smg3p and Smg4p in Caenorhabditis elegans) (Leeds et al. 1991; He and Jacobson 1995; Lee and Culbertson 1995; He and Jacobson 2015). The Upf proteins interact with each other and are considered as the core factors for NMD in eukaryotes (He and Jacobson 2015).

Upf1p is a cytoplasmic RNA helicase with two major functional domains— a cysteine- and histidine-rich (CH) domain at its N-terminal and a helicase domain (Leeds et al. 1992; Czaplinski et al. 1995; Weng et al. 1996; Kadlec et al. 2006). The helicase domain has RNA-binding activity, RNA-dependent ATPase activity and ATP-dependent RNA helicase activity in both yeast and humans (Czaplinski et al. 1995; Weng et al. 1996; Bhattacharya et al. 2000). The CH domain interacts with Upf2p, a scaffold protein that acts as a molecular bridge between Upf1p and Upf3p (He et al. 1997). Upf3p is a basic protein that shuttles between the nucleus and the cytoplasm, and it has two isoforms (a and b) in human cells (Shirley et al. 1998; Lykke- Andersen et al. 2000; Serin et al. 2001). Upf1p is the key regulator of NMD, and its interaction with Upf2p and Upf3p regulates Upf1p activity (Chamieh et al. 2008). It has been shown in yeast that the overexpression of UPF1 can compensate for mutations in UPF2 and UPF3, but not vice versa (Maderazo et al. 2000). Notably, Upf1p also interacts with eRF1 and eRF3 in yeast and humans (Czaplinski et al. 1998; Kashima et al. 2006; Ivanov et al. 2008; Singh et al. 2008).

In addition to the core Upf proteins, NMD in multicellular organisms requires several other proteins, including Smg1p and Smg5-9p (Kervestin and Jacobson 2012; Schweingruber et al. 2013; He and Jacobson 2015; Karousis et al. 2016). The kinase Smg1p forms a complex with Smg8p and

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Smg9p, which catalyses the of Upf1p (Kashima et al. 2006; Yamashita et al. 2009; Yamashita 2013). Smg5p, Smg6p, and Smg7p are involved in the dephosphorylation of Upf1p and the degradation of NMD substrates (Yamashita 2013). Smg5p and Smg7p form a dimer that recruits the CCR4-NOT complex and promotes mRNA deadenylation and decapping (Unterholzner and Izaurralde 2004; Loh et al. 2013; Yamashita 2013). Smg6p promotes the endonucleolytic cleavage of NMD substrates (Huntzinger et al. 2008; Eberle et al. 2009). Notably, Upf1p phosphorylation by a yet undetermined kinase has also been observed in yeast, but it is unclear if phosphorylation regulates Upf1p activity (de Pinto et al. 2004; Wang et al. 2006; Lasalde et al. 2014). In mammalian cells, NMD activation involves the (EJC), which is a protein complex deposited 20-24 nucleotides upstream of an exon-exon junction (Le Hir et al. 2000; Le Hir et al. 2001; Karousis et al. 2016). The observation that EJC associates with Upf3p and Smg6p suggests that EJC serves as an anchor point for NMD factors (Le Hir et al. 2001; Kashima et al. 2010).

NMD models Several different models for the NMD pathway have been proposed (Kervestin and Jacobson 2012; He and Jacobson 2015; Karousis et al. 2016). The major differences between the various models concern the recruitment of Upf1p to the mRNAs and the feature of PTCs that causes NMD activation. It has been believed that Upf1p is recruited preferentially to NMD substrates in a translation-dependent manner, and this hypothesis is supported by evidence from yeast and worms (Johansson et al. 2007; Johns et al. 2007). Recent observations in cancer cells suggest that Upf1p is indiscriminately recruited to both targets and non-targets before translation (Hogg and Goff 2010; Zund et al. 2013; Kurosaki et al. 2014). However, it has been mentioned that phosphorylated human Upf1p binding is specific for NMD substrates and requires mRNA translation (Zund et al. 2013; Kurosaki et al. 2014). Thus, these observations may indicate that the NMD specificity of Upf1p is regulated by its phosphorylation status in higher eukaryotes due to the increased complexity in mRNP and ribosomal structures. For the NMD-initiating feature of PTCs, the EJC model suggests that the EJC has an important role in Upf1p activation. The EJC associates with Upf3p and travels with a mRNA to the cytoplasm, where Upf2p associates with Upf3p and Upf2p-Upf3p activates Upf1p (He and Jacobson 2015). The faux-UTR model suggests that the lack of a certain protein factor that promotes normal translation termination (e.g., the poly(A) binding protein), leads to aberrant translation termination and subsequent NMD activation (He and Jacobson

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2015). Whether it is the presence of (in the EJC model) or the absence of (in the faux-UTR model) certain components in the premature 3’-UTR mRNP, the basic concept is the same; that is the 3’-UTR of a PTC differs from the 3’-UTR of a normal termination codon and that difference triggers the degradation of a PTC-containing transcript by NMD. Notably, the mechanisms of the Upf2p and Upf3p recruitment to the target mRNA is clearer in the EJC model.

Accordingly, a reconciling model for the activation of NMD can be described as follows: upon the translation termination at a PTC, its aberrant 3’-UTR mRNP context leads to the recruitment and/or activation of Upf1p. In yeast, Upf1p recruits the Dcp1p-Dcp2p decapping enzyme via an interaction with Dcp2p (He and Jacobson 1995). After deadenylation-independent decapping by Dcp1p-Dcp2p, the substrate mRNA is subjected to 5’ to 3’ exonucleolytic degradation by Xrn1p (Muhlrad and Parker 1994; He and Jacobson 2001). In addition to the predominant deadenylation-independent 5’ to 3’ degradation, NMD in yeast also involves accelerated deadenylation followed by 3’ to 5’ degradation by the cytoplasmic exosome (Cao and Parker 2003; Mitchell and Tollervey 2003; Takahashi et al. 2003). In human cells, phosphorylated Upf1p recruits Smg5p-Smg7p dimer to the substrate transcript (Ohnishi et al. 2003; Okada-Katsuhata et al. 2012). The dimer recruits the CCR4-NOT deadenylation complex via Smg7p, causing deadenylation of the substrate (Unterholzner and Izaurralde 2004; Loh et al. 2013). Upf1p also recruits Dcp2p to the substrate for decapping, and the transcript is eventually degraded by Xrn1p (Lykke-Andersen 2002; Lejeune et al. 2003). In addition to the described decay mechanism, NMD in humans also involves an endonucleolytic decay pathway that begins with the recruitment of the endonuclease Smg6p to the target transcript by Upf1p (Okada-Katsuhata et al. 2012; Chakrabarti et al. 2014; Nicholson et al. 2014). Smg6p cleaves the substrate in the vicinity of the PTC and generates two fragments; the 5’ fragment is degraded by a cytoplasmic exosome, and the 3’ fragment is degraded by Xrn1p (Huntzinger et al. 2008; Eberle et al. 2009). The truncated polypeptide is degraded via the ubiquitin-proteasome pathway (Takahashi et al. 2008; Kuroha et al. 2009).

NMD inactivation and nonsense suppression Inactivation of the NMD pathway leads to the stabilization and/or accumulation of PTC-containing transcripts (He et al. 1993; Lykke-Andersen et al. 2000; Gatfield et al. 2003). Genome-wide studies in yeast, fly, and human cells have shown that approximately 3-10% of cellular transcripts are

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up-regulated upon inactivation of the NMD pathway (Kervestin and Jacobson 2012). In yeast, NMD inactivation also leads to nonsense suppression (Leeds et al. 1992). One cause of the nonsense suppression phenotype is elevated levels PTC-containing transcripts in NMD-deficient cells (Leeds et al. 1992). Increased translational readthrough of PTCs in NMD mutants also contributes to the nonsense suppression phenotype, which was thought to be caused by a lack of interaction between Upf proteins and release factors (Maderazo et al. 2000; Wang et al. 2001; Keeling et al. 2004). However, Johansson and colleagues provided evidence that the increased readthrough of PTCs is a result of elevated magnesium ion levels in NMD-deficient cells (Johansson and Jacobson 2010). Two Mg2+ importers, Alr1p and Alr2p, have been identified on the plasma membrane in yeast cells (MacDiarmid and Gardner 1998). Mutations in the ALR1 gene counteracts the nonsense suppression phenotype, and its mRNA is targeted by the NMD pathway for degradation (Johansson and Jacobson 2010). One uORF in the 5’-UTR of the ALR1 mRNA triggers the down-regulation of the ALR1 mRNA and protein levels by NMD, and cellular Mg2+ levels are increased in NMD-deficient cells as a result of elevated Alr1p expression (Johansson and Jacobson 2010). Although ALR2 mRNA is also an NMD substrate, Alr2p plays only a minor role in Mg2+ uptake and the alr2Δ allele does not counteract the nonsense suppression phenotype in NMD-deficient cells (MacDiarmid and Gardner 1998; Wachek et al. 2006; Johansson and Jacobson 2010). In summary, NMD inactivation stimulates the readthrough of PTCs as a result of elevated cellular Mg2+ levels, which suggests an indirect effect of the NMD pathway on translational fidelity and Mg2+ homeostasis in yeast.

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RESULTS AND DISCUSSION

Paper I: The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression.

In eukaryotes, transfer RNA (tRNA) precursors are generated by RNA Polymerase III transcription and must pass through a series of processing events that produce mature tRNA molecules (Phizicky and Hopper 2010). Nucleoside modification at different positions in tRNA is an important step of + Ser tRNA maturation. The yeast sup61 gene encodes tRNACGA and is an essential single-copy gene. A sup61-T47:2C mutant expresses an altered Ser form of tRNACGA and was used to screen for genes that are required for the

Ser modification or maturation of tRNACGA in this mutant (Johansson and Bystrom 2002; Johansson and Bystrom 2004). The TAN1 gene that encodes a tRNA-binding protein was identified in this genetic screen and is required 4 4 for the modified nucleoside N -acetylcytidine at position 12 (ac C12) of serine and leucine iso-acceptors (Johansson and Bystrom 2004).

Unexpectedly, the same screen also identified a bud13 mutant that is lethal in combination with the sup61-T47:2C allele. HPLC analyses of nucleosides derived from the total tRNA revealed that the deletion of the BUD13 gene leads to a substantial reduction of the ac4C level in tRNA. The remaining ac4C levels were approximately 5% of those in wild-type cells at 30˚C. Deletion of the BUD13 gene, when combined with the sup61-T47:2C Ser allele also caused a reduction in the tRNACGA levels as well as a severe growth defect at 25˚C.

Bud13p is a subunit of the RES complex, a non-snRNP complex that is associated with the spliceosome (most likely with U2 snRNP) before the first catalytic step of splicing (Dziembowski et al. 2004). We questioned whether the other two RES subunits, Snu17p and Pml1p, affected the formation of ac4C in tRNA. We found that the ac4C levels in snu17Δ cells at 30˚C were similar to those in bud13Δ cells, which is approximately 5% of the wild-type levels. In pml1Δ cells, the ac4C levels were approximately the same as the wild-type levels at 30˚C and approximately 65% of the wild-type levels at 37˚C. The snu17Δ sup61-T47:2C cells showed a similar growth defect as the bud13Δ sup61-T47:2C cells, i.e., they grew slowly at 25˚C and were not viable at 30˚C or 37˚C. The pml1Δ sup61-T47:2C cells did not show growth

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defects at 25˚C and 30˚C, but evidenced a slight synergistic growth defect at 37˚C. In summary, RES mutants show defects in formation of ac4C in tRNA, and the effects of individual mutants on the ac4C levels is correlated with the importance of the respective factor for splicing, i.e., bud13Δ and snu17Δ alleles cause stronger defects than the pml1Δ allele.

Because no indication that RES complex has a direct role in tRNA modification was evident, we hypothesized that the observed decrease of ac4C in the RES mutants could be caused by the inefficient splicing of the pre-mRNA from a gene involved in ac4C formation. The TAN1 gene has a 58 nucleotide (nt) intron at the 5’ of the ORF. Northern analyses of wild-type, bud13Δ, snu17Δ and pml1Δ cells showed the accumulation of TAN1 pre- mRNA in all three RES mutants and no spliced TAN1 mRNA was detected in the bud13Δ and snu17Δ strains at 30˚C. The splicing defect in the pml1Δ strain was not as strong as in the other two RES mutants at 30˚C, but became stronger at 37˚C, which is consistent with the observed reduction of ac4C in the pml1Δ cells at different temperatures. Western blotting of strains that expressed three tandem influenza virus haemagglutinin epitopes (3HA) tagged Tan1p confirmed that the Tan1p levels were lower in pml1Δ cells and Tan1p was not detectable in bud13Δ and snu17Δ cells. An HPLC analysis of the tan1Δ bud13Δ strain that contained an intron-less TAN1 gene on a low- copy plasmid showed restored ac4C levels in tRNA, which confirmed that the RES-dependent regulation of TAN1 pre-mRNA splicing promotes the formation of ac4C in tRNA.

The total abundance of TAN1 transcripts was lower in the RES-deficient cells compared with the wild-type cells. Because the RES complex has a nuclear retention function and unspliced pre-mRNAs are usually targeted by the NMD pathway for degradation, the decreased abundance of TAN1 transcripts suggested that inefficiently spliced TAN1 pre-mRNA might be exported to the cytoplasm and degraded by the NMD pathway, (He et al. 1993; Dziembowski et al. 2004). To directly evaluate this possibility, we combined the upf1Δ allele with individual RES mutants and analysed the total abundance of TAN1 transcripts. The results showed that the abundance of TAN1 transcripts was restored when the NMD pathway was inactivated in the RES-deficient cells. Decay rate measurements of the TAN1 pre-mRNA confirmed it is an NMD substrate in the RES mutants.

The RES complex has been previously suggested as particularly important for the efficient splicing of pre-mRNAs containing non-consensus 5’ splice site sequences (Dziembowski et al. 2004; Spingola et al. 2004;

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Scherrer and Spingola 2006). However, the TAN1 intron has the canonical 5’ splice site sequence GUAUGU (Spingola et al. 1999), indicating that the 5’ splice site sequence is not likely the feature that induces RES-dependency. We probed control transcripts CYH2, RPL25 and GOT1 to assess the effect of the RES complex on their splicing efficiency. These pre-mRNAs all have the canonical 5’ splice site sequence and their introns have different lengths, but the splicing and nuclear retention of these transcripts were largely unaffected in the RES-deficient cells. To test if the intron promotes a requirement for the RES complex, we replaced the endogenous TAN1 intron sequence with the intron sequence of the GOT1 or RPL25 gene. The hybrid TAN1 pre-mRNAs were efficiently spliced in the RES-deficient cells compared to the original TAN1 pre-mRNA. We also replaced the endogenous GOT1 intron sequence, which is similar to TAN1 intron in length, with the intron sequence from the TAN1 gene. The GOT1 pre- mRNAs that contain the TAN1 intron became inefficiently spliced in the RES-deficient cells, and accumulated in bud13Δ upf1Δ cells, which is quite similar to the endogenous TAN1 pre-mRNA. Accordingly, the TAN1 intron is sufficient to mediate the RES-regulated splicing of its own pre-mRNA.

Both the TAN1 intron and the GOT1 intron have consensus sequences for the 5’ splice site, the branchpoint and the 3’ splice site. To further investigate the intronic feature that potentially causes a requirement for the RES complex, we replaced the sequence between the endogenous GOT1 5’ splice site and the branchpoint with the corresponding sequence from the TAN1 intron. We also replaced the sequence between the endogenous GOT1 branchpoint and the 3’ splice site with the corresponding sequence from the TAN1 intron. Northern blot analyses of the strains that contain these hybrid GOT1 alleles showed that the GOT1 pre-mRNA with the sequence between the 5’ splice site and the branchpoint substituted was inefficiently spliced in the bud13Δ cells, and accumulated in the bud13Δ upf1Δ cells, suggesting that the 30 nucleotide sequence between the TAN1 5’ splice site and the branchpoint is sufficient to elicit the requirement for the RES complex for efficient splicing.

The abundance of modified nucleosides in tRNA is affected under stress conditions (Chan et al. 2010). The splicing efficiency of the pre-mRNAs can also be affected by environmental stress (Bergkessel et al. 2011). Thus, it is possible that the formation of ac4C in tRNA is controlled via the splicing regulation of TAN1 pre-mRNA under some conditions.

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Paper II: The pre-mRNA retention and splicing complex controls expression of the Mediator subunit Med20

Yeast cells lacking any RES factors show growth defects that are correlated with their individual importance for splicing, and the growth defects are exacerbated at elevated temperature (Dziembowski et al. 2004). The cause of such a Ts phenotype was not known, and we postulated that the inefficient splicing of a certain transcript results in the Ts phenotype in the RES-deficient cells. We did a gene-dosage suppressor screen to address this question, and searched for genes that suppress the Ts phenotype of the bud13Δ cells at 37˚C. The reason for using the bud13Δ strain is that it has a stronger Ts phenotype than the snu17Δ and pml1Δ strains.

We identified several gene-dosage suppressors from the screen, one of which, MED20, is an intron-containing gene. The MED20 gene, also known as SRB2, contains a 101 nucleotide intron on the 5’ side of the ORF and encodes a subunit of the yeast Mediator complex (Koleske et al. 1992; Thompson et al. 1993; Kim et al. 1994; Lariviere et al. 2006). The Mediator complex is an important co-regulator of RNA Pol II transcription, and Med20p is required for basal level transcription in vitro (Thompson et al. 1993). Med20p forms the head module of the Mediator with Med8p and Med18p, and med20Δ cells exhibit a growth defect that is exacerbated at elevated temperature (Nonet and Young 1989; Lariviere et al. 2006). We transformed individual RES mutants with a high-copy plasmid that contained a wild-type MED20 gene. The results showed that the overexpression of the MED20 gene can suppress the Ts phenotype of the bud13Δ cells and the snu17Δ cells, but no apparent suppression was observed in pml1Δ cells.

Northern blot analyses revealed that the MED20 pre-mRNA is poorly spliced in the RES-deficient cells. At 30˚C, the level of spliced mRNA is approximately 20% in bud13Δ cells and snu17Δ cells compared to wild-type cells, and approximately 50% in pml1Δ cells. At 37˚C, the level of spliced mRNA is similar to 30˚C in bud13Δ cells and snu17Δ cells, but slightly decreased in pml1Δ cells. To assess if the spliced MED20 mRNA levels correlate with Med20p levels in the RES-deficient cells, we did western blot assay in strains that expressed 3HA tagged Med20p. The western results were in good agreement with the northern data, suggesting that inefficient splicing of the MED20 pre-mRNA results in decreased Med20p levels which subsequently cause the Ts growth phenotype of the bud13Δ and snu17Δ cells. To confirm that the observed Ts phenotype in bud13Δ and snu17Δ mutants is due to the inefficient splicing of the MED20 pre-mRNA, we

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removed the endogenous MED20 intron sequence in the RES-deficient cells so that the expression of the Med20p became independent of splicing and the RES complex. The MED20 mRNA levels were restored in the RES mutants that contained an intron-less MED20 allele, and the Ts growth defects were largely suppressed in the bud13Δ and snu17Δ mutants.

Because the RES complex promotes pre-mRNA nuclear retention and the total abundance of the MED20 transcripts was lower in the RES-deficient cells, we suspected that the MED20 pre-mRNA might be exported to the cytoplasm and targeted by the NMD pathway for degradation. Northern blot analyses showed the accumulation of the MED20 pre-mRNA in the RES mutants when NMD is inactivated, suggesting that the MED20 pre-mRNA is an NMD substrate. However, the low abundance of the MED20 pre-mRNA in the RES mutants made it difficult to determine the effect of NMD inactivation on the stability of the MED20 pre-mRNA.

Sequence alignment of the TAN1 and MED20 introns failed to identify any shared features that could potentially trigger RES-dependent splicing. The MED20 intron has the canonical sequence for the 5’ splice site, which suggests that the RES complex is not, as previously proposed, specific for the efficient splicing of transcripts with a weak 5’ splice site sequence (Dziembowski et al. 2004). However, the MED20 intron does have a non- canonical branchpoint sequence UGCUAAC. To assess if the non-canonical branchpoint is the cause of the RES-dependency, we changed the endogenous MED20 branchpoint to the consensus sequence. This change failed to completely restore the splicing efficiency for MED20 pre-mRNA, implying that other features that cause RES-dependency exist. The MED20 intron is similar to the GOT1 intron in size, so we replaced the endogenous MED20 intron sequence with the GOT1 intron sequence. The results showed that in the RES-deficient cells, splicing of the MED20 pre-mRNA with a GOT1 intron was even more efficient than with the consensus branchpoint sequence. We also replaced the endogenous GOT1 intron with the intron sequence from MED20, but we only observed a lower abundance of the spliced new GOT1 transcript in the RES-deficient cells. No accumulation of the MED20 intron-containing GOT1 pre-mRNA was shown in the upf1Δbud13Δ cells. These results indicated that the cis-element responsible for the RES-regulated splicing of MED20 is not limited to the MED20 intron, and may differ from the one for TAN1 splicing. To further investigate the element required for RES-dependency in the MED20 pre- mRNA, we replaced both the GOT1 exon 1 and intron with the

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corresponding sequences from MED20. Our northern results showed that this MED20-GOT1 pre-mRNA became inefficiently spliced and accumulated in both the bud13Δ and the upf1Δbud13Δ cells. We did not observe the effect of NMD inactivation, that is, the upf1Δbud13Δ strain did not show increased accumulation of the MED20-GOT1 pre-mRNA compared to the bud13Δ strain. This is due to a lack of a PTC in the pre-mRNA. Taken together, the RES-dependent splicing regulation of the MED20 pre-mRNA requires both intronic and exonic elements, which is different from the element of the TAN1 pre-mRNA, indicating that the cis-element that induces RES-dependency may not be a simple recognition site.

The overexpression of MED20, which encodes a Mediator complex subunit, suppresses the Ts growth phenotype in the bud13Δ cells and the snu17Δ cells. This finding suggests that the Ts phenotype in the bud13Δ and snu17Δ mutants is largely attributable to defects in the Mediator functionality (Lariviere et al. 2006). We also found that inactivation of the NMD pathway suppressed the Ts phenotype of the bud13Δ cells and the snu17Δ cells, which is likely due to a slight increase of the MED20 mRNA upon NMD inactivation at 37˚C. However, the suppression of the growth defect of the bud13Δ cells at 30˚C by NMD inactivation indicates that the altered expression of other genes may also contribute to the suppression by the upf1Δ allele.

The RES complex was initially identified as a non-snRNP complex that is required for the efficient splicing of transcripts encompassing a suboptimal 5’ splice site sequence in its intron (Dziembowski et al. 2004). This notion is supported by the identification of AMA1 and MER2 pre-mRNAs as RES targets (Scherrer and Spingola 2006). In contrast, the pre-mRNAs of MATa1, TAN1 and MED20 contain the consensus 5’ splice site sequence, and require the RES complex for the efficient splicing (Schmidlin et al. 2008; Tuo et al. 2012). These findings suggest that a suboptimal 5’ splice site is not the defining feature for RES-dependent splicing. Moreover, alignment of the introns from the identified RES targets failed to produce significant sequence or structural similarity. Therefore, the preference for the RES complex to enhance the splicing of a pre-mRNA is still unclear. It is possible that the RES complex is required when the splicing of a transcript is intrinsically inefficient.

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Paper III: The nonsense-mediated mRNA decay pathway controls the expression of magnesium transporters in Saccharomyces cerevisiae

NMD inactivation in yeast leads to a nonsense suppression phenotype, which is partially attributable to the increased translational readthrough of PTCs in NMD-deficient cells (Maderazo et al. 2000; Wang et al. 2001; Keeling et al. 2004). The increased readthrough of PTCs has been demonstrated to result from higher cellular Mg2+ levels, which are caused by the up-regulation of Alr1p expression (Johansson and Jacobson 2010). The ALR1 mRNA is targeted by the NMD pathway for rapid degradation. Although the DNA sequence upstream of the ALR1 ORF contains multiple potential uORFs (uORF1, 2A/B and 3), transcripts carrying uORF1 and uORF2A/B are in low abundance and do not appear to contribute to the NMD-regulation of Alr1p expression (Johansson and Jacobson 2010). It is unclear whether the ribosome reinitiates at the ALR1 main ORF after termination at uORF3, or the scanning ribosome bypasses uORF3 and initiates at the main ORF. To address this question, we developed a luciferase reporter system in which a renilla luciferase (rluc) gene is the reporter and a firefly luciferase (fluc) gene serves as an internal control. The sequence of interest can be inserted in front of the rluc gene to study the effect of a specific and the 5’-UTR sequence on gene expression. In this study, we cloned DNA sequences containing different mutations in ALR1 5’-UTR into the reporter system. The results showed that the relative rluc expression level was significantly decreased as the distance between the uORF3 and the main ORF decreased, suggesting that the ribosome reinitiates at the downstream main ORF after translating uORF3 (Kozak 1987b; Abastado et al. 1991). To test whether differences in mRNA abundance contribute to the differences in relative rluc expression, we repeated the experiments in upf1Δ cells. The results were in good agreement with those from wild-type cells, conforming that ribosomes mostly reinitiate translation at the ALR1 main ORF after translation termination at uORF3.

We conclude that the majority of scanning ribosomes initiate translation of the ALR1 uORF3, terminate at the uORF3 stop codon, and reinitiate translation at the downstream main ORF. It has been shown that Alr1p expression is increased during Mg2+ starvation with no change in the ALR1 mRNA level (Lim et al. 2011). Thus, it is possible that the translation initiation efficiency of the ALR1 uORF3 is controlled to regulate the expression level of the ALR1 gene under Mg2+ starvation conditions. In this

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study, we grew yeast cells in a Mg2+-sufficient medium, so the effects of Mg2+ on the expression of Alr1p remain to be tested.

The NMD pathway is suggested to control Mg2+ homeostasis by down- regulating the expression of the ALR1 and ALR2 mRNAs (Johansson and Jacobson 2010). Yeast cells contain several organellar Mg2+ transporters, including the vacuolar Mg2+ exporter Mnr2p, the mitochondrial Mg2+ importer Mrs2p/Lpe10p and the mitochondrial Mg2+ exporter Mme1p. The MNR2 5’- UTR is predicted to encompass 4 uORFs, and one of them is potentially translated (Miura et al. 2006; Ingolia et al. 2009). This suggests that MNR2 mRNA is a potential NMD substrate. To assess if MNR2 mRNA, as well as MRS2 and MME1 mRNAs, are targeted by the NMD pathway for degradation, we measured their mRNA abundance and mRNA decay rates in NMD-deficient cells and NMD-sufficient cells. Transcripts from LPE10 were not included because Lpe10p serves only as the regulator of Mrs2p (Sponder et al. 2010). The MNR2 mRNA showed an approximate 40% accumulation upon NMD inactivation, while MRS2 and MME1 mRNAs increased by approximately 50% and approximately 30% respectively in NMD-deficient cells. The MNR2 and MME1 mRNAs were stabilized in NMD- deficient cells, while the decay rate of MRS2 mRNA is not affected by NMD inactivation. In summary, these results suggested that MNR2 and MME1 mRNAs are NMD substrates.

The lack of Alr1p leads to a decreased translational readthrough of PTCs and counteracts the nonsense suppression phenotype in NMD mutants as a result of lower cellular Mg2+ levels (Johansson and Jacobson 2010). To test if a lack of any organellar Mg2+ transporter can counteract the nonsense suppression phenotype, we constructed strains that contained the mnr2Δ, mrs2Δ, and mme1Δ alleles separately and combined these alleles with the upf1Δ allele. These strains also contain leu2-2 (UGA) and can1-100 (UAA) nonsense alleles. In upf1Δ cells, stop codons of these nonsense alleles are read by near-cognate tRNAs, generating full length Leu2p and Can1p. This can be indicated by growth on medium without leucine (SC-leu medium) and sensitivity to canavanine (SC-arg+can medium) (Weng et al. 1996; Maderazo et al. 2000). The results suggested that a MNR2 deletion counteracts the nonsense suppression of leu2-2 and can1-100, and the mrs2Δ allele counteracts the nonsense suppression of the leu2-2 allele. Deletion of MME1 did not have any effect. We further determined if the observed effects on nonsense suppression are due to a decrease in the translational readthrough of PTCs. Data from a dual luciferase reporter

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system (Keeling et al. 2004) showed that a lack of Mnr2p or Mrs2p slightly decreased the translational readthrough of the stop codons. However, these effects are not as significant as the effect of the lack of Alr1p.

A previous study showed that organellar Mg2+ can be exported to the cytoplasm via Mnr2p and Mme1p under Mg2+ starvation conditions (Pisat et al. 2009; Cui et al. 2015). Thus, our finding that MNR2 and MME1 mRNAs are targeted by the NMD pathway suggests that the NMD pathway specifically degrades transcripts of Mg2+ transporters (i.e., Alr1p, Mnr2p and Mme1p) that are responsible for mediating the Mg2+ influx into the cytoplasm. A lack of Mnr2p or Mrs2p had minor effects on the readthrough of stop codons, and a lack of Mme1p had no detectable effect. The different effects of the alr1Δ, mnr2Δ, and mme1Δ alleles on the readthrough of stop codons may simply reflect their individual capacity to modulate the cytoplasmic Mg2+ concentration. It is possible that the effects of the mnr2Δ and mme1Δ alleles are masked by Alr1p, and their effects on the readthrough of stop codons would be more substantial under Mg2+ starvation conditions. Accordingly, we propose that the NMD pathway maintains the cytoplasmic Mg2+ concentration by regulating the expression of ALR1, MNR2 and MME1.

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CONCLUSIONS

1) The RES complex controls the formation of ac4C in the tRNAs by promoting splicing of the TAN1 pre-mRNA.

2) The Ts phenotype in the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA.

3) Unspliced TAN1 and MED20 pre-mRNAs that enter the cytoplasm are degraded by the NMD pathway.

4) The cis-acting elements that mediate RES-dependency are present in both exon and intron sequences.

5) Translation initiation at the ALR1 main ORF is largely dependent on a reinitiation mechanism.

6) The NMD pathway targets the MNR2 and MME1 mRNAs for degradation.

7) A lack of the organellar Mg2+ transporters Mnr2p or Mrs2p causes a decrease in the translational readthrough of stop codons.

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ACKNOWLEDGEMENTS

First of all, I would like to express my sincere gratitude to my supervisor, Dr. Marcus J.O. Johansson. He is the one who provided me with the opportunity to be scientifically trained in the beginning. I would like to thank him for his generous help both inside and outside academia.

I would also like to thank Fu, Hao, Tony, Hasan (and his family) and Anders (thanks for showing me your LP collection) from group ABy, Mridula, Susanne (and her family) and Dr. Nissan (thanks for giving me all the laughter) from group TN, and Gunilla and Glenn (thanks for your comments and encouragement) from group GB. I wouId like to thank colleagues that attended the beer corner for all the bizzare discussions. I want to thank Sa Chen and Hairu Yang for their kindness. I would like to thank Dr. Jie Song and Mr. Fu Xu for constructive comments on this thesis. I also would like to thank our collaborators Dr. Changchun Chen and Dr. Vasili Hauryliuk. I would also like to thank all former members from group Smap (especially Mr. Kimura Takuya) for their excellent work. Dishwashing, media, administration and cleaning personnel have provided valuable services. I would like to thank Mr. Johnny Stenman for taking care of all my deliveries (both scientific and non-scientific). Finally, I would like to thank my parents (and my mormor) for their unconditional support.

鸣谢 感谢曾在瑞典帮助过我的每一个人，不论你是否已经离开瑞典。虽然由于 时间的原因我无法在此一一写下你们的名字，但是请相信并接受我由衷的 感谢。 此外，我想借此机会感谢远在国内却一直在支持我的朋友和亲人们。 康汝滢同志，每次回到天津都能受到你一如既往的热情款待，我非常感恩。 刘博宁同学，每次回国你都想方设法带我去欣赏各类文艺演出，从话剧， 肢体剧到钢琴协奏，芭蕾舞，使我这个远在海外文化生活贫瘠的普通青年 能够阶段性地向文艺青年的这条不归路前进。 我还要感谢我的两个舅舅，一个姨，还有八旬有余的姥姥，从我未出襁褓 之时就沉浸在你们亲情的温暖中。不能经常回内蒙古去看望你们，我的内 心充满歉意。

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最后我要谢谢我的爸爸妈妈，感谢你们这七年多来无条件的支持。儿子不 孝，八个春节没回家过了，按以前的说法抗战都胜利了。离开家才切身体 会到什么是每逢佳节必思亲，何谓行人无限秋风思。我争取今后能常回家 看看。

感謝の言葉

天津に居た頃 裏千家（天津）のお茶の先生たちから 様々なご指導を頂 きました。この際に 先生たちへ心から感謝の気持ちを申し上げます。高 妻先生、最初から長い間にいろいろ教えていただきました 「ありがとう」 しか言えません。昔はよく言えませんでしたが この卒業論文で先生に感 謝致します。坂井先生、天津も大阪もお世話になりました。去年の稽古の 御蔭で昔の気持ちを少し覚えていました。

先輩の宮原さんと久下さん、友人として いろいろありがとうございま す。お茶もお酒もまた一緒に飲みましょう。

俳優の木村拓哉さんと松たか子さん、いろいろな役の主演 ありがとう 面白かった。

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