Mrna Degradation Factors As Regulators of the Gene Expression in Saccharomyces Cerevisiae

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS, New Series No 1844, ISSN: 0346-6612, ISBN: 978-91-7601-560-5

mRNA degradation factors as regulators of the gene expression in Saccharomyces cerevisiae

Mridula Muppavarapu

Department of Molecular Biology Umeå University, SE-90187 Umeå 2016

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Copyright© Mridula Muppavarapu ISBN: 978-91-7601-560-5 New series nr: 1844 ISSN: 0346-6612 Cover Picture: Schematic representation of a model of mRNA degradation. Electronic version available at http://umu.diva-portal.org/ Printed by: UmU-tryckservice, Umeå University Umeå, Sweden. 2016

To my Family

"We are kept from our goal, not by obstacles, but by a clear path to a lesser goal." ~ Bhagavad Gita

Table of Contents

Table of Contents i Abstract iii Publications included in this thesis v Abbreviations vi Introduction 1 1. Messenger RNA Biogenesis 2 2. Translation of mRNA 3 3. General cytoplasmic mRNA decay 5 3.1. Decapping-dependent pathway 7 3.2. Exosome-mediated pathway 9 4. Quality Control Mechanisms 10 4.1. Defects in mRNA biogenesis and processing 10 4.1.1. 5’ cap structure 10 4.1.2. Pre-mRNA splicing 11 4.1.3. 3’ end formation 14 4.2. Defects in mRNA due to genetic mutation 14 4.2.1. Nonsense-mediated mRNA decay 14 4.2.2. Non-stop decay 16 4.2.3. No-go decay 17 5. Regulation of gene expression by mRNA decay factors 18 5.1. Modulation of Transcription by mRNA decay factors 22 5.2. Role of mRNA decay factors in Translation 24 5.3. Ribosome Biogenesis and mRNA decay factors 26 5.4. P bodies and mRNA degradation 29

i Aims of the thesis 33 Paper I 33 Paper II 33 Paper III 33 Results and discussion 34 Paper I 34 Paper II 38 Paper III 42 Conclusions 43 Paper I 43 Paper II 43 Paper III 44 Acknowledgements 45 References 48 Articles I-III 67

ii Abstract

Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression. First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle. Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre- mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay. Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that

iii paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability. Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression. In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.

iv Publications included in this thesis

PaperI

Mridula Muppavarapu, Susanne Huch and Tracy Nissan, The cytoplasmic mRNA degradation factor Pat1 is required for ribosomal RNA processing. 2016. RNA Biol. 13(4), 455–465.

PaperII

Susanne Huch, Maren Müller, Mridula Muppavarapu, Jessie Gommlich, Vidya Balagopal, Tracy Nissan, The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae. 2016. Biol Open. 5(10): 1388-1399.

PaperIII

Susanne Huch, Jessie Gommlich, Mridula Muppavarapu, Carla Beckham and Tracy Nissan, Membrane-association of mRNA decapping factors is independent of stress in budding yeast. 2016. Sci Rep. 6, 25477.

v Abbreviations

A-site Aminoacyl site on the ribosome E-site Exit site on the ribosome ETS External transcribed spacers ITS Internal transcribed spacers LLR motif Leucine-rich repeat motif LSm Like Sm m7G cap 7-methylguanosine cap m7GDP cap 7-methylguanosine diphosphate cap m7GMP cap 7-methylguanosine monophosphate cap mRNA Messenger RNA mRNP Messenger RNA-Protein Complex NGD No-go Decay NMD Nonsense-mediated Decay NSD Nonstop Decay P body Processing Body P-site Peptidyl site on the ribosome PIC Pre-initiation complex poly(A) Poly adenosine PTC Pre-termination codon rDNA Ribosomal DNA RNA pol I RNA polymerase I RNA pol II RNA polymerase II RNA pol III RNA polymerase III RPs Ribosomal Proteins

vi rRNA Ribosomal RNA S Svedburg Units snoRNA Small nucleolar RNA snRNA Small nuclear RNA TFs Transcriptional factors TRAMP complex Trf4/Air2/Mtr4p Polyadenylation Complex tRNA Transfer RNA UAS Upstream activating sequence UTR Untranslated regions

vii

Introduction

Control of eukaryotic gene expression occurs at numerous levels. The multi-step process of gene expression begins in the nucleus with the transcription of pre-RNA from DNA by RNA polymerases, which then undergo co-transcriptional and post-transcriptional modifications to become a mature RNA. The pre-RNAs that are transcribed from the non-coding genes, after modifications, become functional RNAs, e.g., ribosomal RNA (rRNA) and tRNA. On the other hand, the pre-RNAs transcribed from coding genes, also known as pre-messenger RNAs or pre-mRNAs, after modifications, are transported into the cytoplasm as mature mRNA for translation by ribosomes into proteins, the final gene product. The life of RNA begins with its transcription, ends with its degradation. RNA degradation primarily facilitates the modulation of gene expression by regulating the levels of mRNA according to the needs of the cell (Parker 2012). Secondly, it acts as a surveillance system that promotes the removal of undesirable or aberrant mRNA that would otherwise translate into defective proteins (Fasken and Corbett 2005; Houseley et al. 2006; Doma and Parker 2007; Parker 2012). Thirdly, it is also required for processing and maturation of pre-RNAs for example rRNA processing (discussed later). Lastly, it facilitates the removal of the byproducts of gene expression like excised introns and rRNA spacers (Houseley et al. 2006; Doma and Parker 2007; Parker 2012).

1. Messenger RNA Biogenesis

Messenger RNA synthesis begins in the nucleus with its transcription as pre-mRNA by RNA polymerase II. Transcription is the first step in the process of gene expression. It is a complex process, orchestrated by several multi-protein complexes involving ~100 individual proteins. In yeast, transcription begins when the co-activators connect with the transcription factors (TFs) at the regulatory DNA elements called upstream activating sequences (UAS) and recruit general transcription factors to the core promoter which then recruits RNA pol II complex to the promoter forming a transcription unit. After dramatic conformational changes, RNA pol II along with other TFs begins to scan for the start site. Once the start site is recognized, RNA pol II together with transcriptional elongation factors begins the synthesis of the pre-mRNA transcript (Hahn and Young 2011).

Co-transcriptionally, RNA pol II, and the associated transcriptional factors facilitate pre-mRNA processing starting with the addition of a 7-methylguanosine (m7G) cap at its 5’ end, a process known as capping (Rasmussen and Lis 1993). After capping, the intervening regions of the pre-mRNA, known as introns that do not become part of the functional protein are removed through a process called splicing and the remaining sequences, called as exons, are joined together. Splicing can occur co-transcriptionally or post-transcriptionally (Bernstein and Toth 2012). Following co-transcriptional splicing, when the RNA pol II reaches the 3’ end of the transcript, the 3’ end of the mRNA also undergoes processing. Elements at the 3’ end of the

mRNA determine the site where it is cleaved and polyadenylated by the poly(A) polymerase, Pap1 (Yonaha and Proudfoot 2000; Bernstein and Toth 2012). In yeast, the average polyadenylated (poly(A)) tail length is ~55-90 nucleotides (Keller 1995; Colgan and Manley 1997; Brown and Sachs 1998; Wahle and Kühn 2001). Many factors involved in 3’ end processing interact with the transcription unit and facilitate transcription termination (McCracken et al. 1997; Tora et al. 1997; Parker 2012). After successful processing, mature mRNA is exported to the cytoplasm in a process coupled to transcription for translation into proteins.

2. Translation of mRNA

Translation, the second major step in the gene expression, is the process by which a protein is synthesized from the information present in mRNA. During translation, the sequence of the mRNA that relates the sequence in the DNA to the proteins is read according to the genetic code. The genetic code is a set of rules that define the sequence of nucleotide triplets called codons, each of which corresponds to a particular amino acid or stop signal.

Translation occurs on the ribosome that is composed of a small (40S) and a large (60S) subunit joined together to form the complete 80S ribosome. Translational initiation is the most complex and highly regulated step of protein synthesis. The Met-tRNAiMet along with many translational initiation factors, bind to the 40S ribosomal subunit to form the 43S pre-initiation complex (PIC). The 43S PIC binds to

the mRNA that bears eIF4E (the cap-binding protein) at the 5’ m7G cap, poly(A) binding protein (Pab1) bound to the poly(A) tail. eIF4G along with eIF4A and eIF4B is believed to prepare the mRNA for binding to 43S PIC, to form 48S PIC. After binding the mRNA at the 5’ end, the 48S complex scans along the mRNA searching for the AUG codon in 5’-3’ direction. Upon AUG recognition, several bound initiation factors are released, and the ribosomal complex undergoes conformational changes. After that, 60S subunit joins to the AUG- bound PIC and the complete 80S complex with Met-tRNAiMet in the P-site that is ready for translational elongation is formed (Dever et al. 2016).

During translational elongation, the amino acyl-tRNA carrying the next amino acid is delivered to the cognate codon in the A-site that base pairs with the mRNA. There after, the carboxyl end of the peptide chain is released from the tRNA at the P-site and joined to the free amino acid bound to the tRNA in the A-site forming a peptide bond in a process catalyzed by the ribosome. After that, the A-site peptidyl-tRNA translocates to the P-site and the now deacylated tRNA in the P-site moves to the E-site and the A-site is empty for the next amino acyl-tRNA. The elongation continues until the ribosome reaches a stop codon (UAA, UAG, and UGA) which is not recognized by a tRNA and do not specify an amino acid. The release factors bind the ribosome with the empty A-site to facilitate the termination and release of the polypeptide chain. After translation, when the poly(A) tail shortens or the mRNA is no longer required, it is targeted for general mRNA decay.

3. General cytoplasmic mRNA decay

The pathways of general cytoplasmic mRNA degradation are the well- studied pathways of RNA decay in Saccharomyces cerevisiae. mRNA has two important structures, the m7G cap at the 5’ end and poly(A) tail at the 3’ end. These two structures promote translation and protect the mRNA from degradation. In yeast, general mRNA degradation begins with the shortening of the poly(A) tail also known as deadenylation. After deadenylation, mRNA can be degraded either from the 5’ to 3’ direction through the 5’ to 3’ mRNA decay pathway (decapping-dependent pathway) or from the 3’ to 5’ direction via the 3’ to 5’ mRNA decay pathway (exosome-mediated pathway) (Figure 1) (Parker and Song 2004; Houseley et al. 2006; Doma and Parker 2007; Parker 2012; Das and Das 2013; Huch and Nissan 2014).

Shortening of the poly(A) tail is the first and the rate limiting step in both the pathways of general cytoplasmic mRNA degradation. Deadenylases require a 3’OH to catalyze deadenylation and release AMP during the process. The multifunctional Ccr4-Pop2-Not and the Pan2-Pan3 are the two deadenylase complexes that catalyze poly(A) tail shortening (Rasmussen and Lis 1993; Collart 2003). Deletion of Ccr4 together with Pan2 completely blocks deadenylation (Chen et al. 2002; Bernstein and Toth 2012). The Ccr4-Pop2-Not deadenylase complex comprises of two exonucleases, Ccr4 and Pop2.

m7Gppp AUG STOP Poly(A)

CCR4-NOT complex (Ccr4, Caf1,Not1-5, Caf40,Caf30) PAN2-PAN3 complex

Pan3 Pan2 Deadenylation Deadenylation Caf1

Ccr4 Pan3 Pan3 m7Gppp AUG STOP Oligo(A)

Exosome mediated

Dcp1/Dcp2complex Exosome complex ( Rrp4, Decapping activators pathway Rrp40,41,42,43,Rrp45, (Pat1, Lsm1-7, Dhh1, Rrp46,Mtr3, Csl4, Dis3) Edc3, Scd6) Decapping Cofactors: Ski1,Ski2,Ski4, Ski8,Ski7 dependent pathway Exosome Dcp1 Dcp2 p AUG STOP Oligo(A) m7Gppp AUG STOP Cap hydrolysis

Xrn1 Dcs1 Exonuclease Exonuclease

Dcs1 pp Xrn1 STOP Oligo(A)

Figure 1. Pathways of general mRNA degradation in Saccharomyces cerevisiae

Although Ccr4 is the main deadenylase of the complex, the strains lacking Pop2 are defective in deadenylation (Yonaha and Proudfoot 2000; Tucker et al. 2001; Daugeron 2001; Tucker et al. 2002; Parker and Song 2004; Bernstein and Toth 2012). Although, Pop2/Caf1 has a catalytic site, a mutation to it does not affect deadenylase activity of the complex and overexpression of Ccr4 suppresses the deadenylation defect (Keller 1995; Colgan and Manley 1997; Brown and Sachs 1998; Wahle and Kühn 2001; Tucker et al. 2002; Thore et al. 2003; Viswanathan et al. 2004). Pop2 is believed to recruit Ccr4 to the large Ccr4-Pop2-Not complex through the leucine-rich repeat motif (LLR)

and thus promote the function of the Ccr4 (Clark et al. 2004). Apart from the two exonucleases, this complex also contains seven other subunits, Not1, Not2, Not3, Not4, Not5, Caf40 and Caf130 (Chen et al. 2001; Denis and Chen 2003; Bhaskar et al. 2013). Although they do not possess any clear function, they are suggested to play regulatory roles in other functions of Ccr4-Pop2-Not complex (Tucker et al. 2002; Deluen et al. 2002; Swanson et al. 2003; Qiu et al. 2004; Kruk et al. 2011a).

Pan2-Pan3 is the second deadenylase complex in yeast. Pan2, together with the homodimer of Pan3, forms a Pan2-Pan3 heterotrimeric complex (Wahle and Winkler 2013). The 3’ to 5’ exonuclease Pan2, is the catalytic subunit of this complex that shortens the poly(A) tail from ~95 to ~65 residues. The activity of Pan2-Pan3 complex depends on Pab1 (Boeck et al. 1996). A model that describes the temporal dynamics of the deadenylation is that the Pan2-Pan3 complex facilitates the shortening of the 3’ most region of the poly(A) tail to ~65 nucleotides, after which Ccr4-Pop2-Not complex takes over and slowly degrades the poly(A) tail (Brown and Sachs 1998; Tucker et al. 2001). Once the mRNA becomes oligoadenylated (10-12 nucleotides), it can be degraded either by decapping-dependent pathway or exosome-mediated pathway.

3.1. Decapping-dependent pathway

Degradation of mRNA by the decapping-dependent pathway is the major pathway of mRNA degradation in yeast (Balagopal et al. 2012).

It starts with the removal of the 5’ m7G cap by the Dcp1-Dcp2 complex (Figure 1). Dcp2 is the catalytic subunit of the decapping enzyme complex that hydrolyzes the 5’ m7G cap to release m7GDP and 5’ monophosphate mRNA (She et al. 2008; Li and Kiledjian 2010). The Dcp1-Dcp2 enzyme complex has a preference for longer mRNAs (Steiger et al. 2003). The first step in the process of mRNA decapping is translational repression (Parker 2012; Radhakrishnan and Green 2016). After which the decapping enzyme complex replaces the translational initiation factors at the cap structure. The decapping enhancers can then activate Dcp2 to catalyze the cap removal (Parker 2012). The coenzyme Dcp1 is the chief promoter of the catalytic activity of Dcp2, but there are many decapping activators that can regulate decapping (Coller and Parker 2005; She et al. 2008; Deshmukh et al. 2008; Balagopal and Parker 2009; Valkov et al. 2016).

There are two types of decapping activators 1) factors that directly activate the decapping enzyme and 2) factors that repress translation thereby activating the mRNA decapping (Nissan et al. 2010). Proteins like Edc1, Edc2, Edc3, Dcp1, and Pat1 have been shown to stimulate decapping enzyme directly (Schwartz et al. 2003; She et al. 2008; Harigaya et al. 2010; Nissan et al. 2010; Borja et al. 2011; Parker 2012). On the other hand, Pat1, Dhh1, Scd6, Stm1 and Lsm1-7 inhibit translation both in vitro and in vivo to enhance decapping (Pilkington and Parker 2008; Balagopal and Parker 2008; Nissan et al. 2010; Rajyaguru et al. 2012). Pat1 is the only known protein that can directly activate decapping enzyme and also repress translation, which

will, in turn, lead to activation of the decapping enzyme. Pat1 is a multifunctional protein that forms a complex with seven Sm-like proteins (Lsm) 1-7, to regulate mRNA decay (Tharun and Parker 2001; Chowdhury et al. 2007; Chowdhury and Tharun 2008; Tharun 2009; Nissan et al. 2010; Marnef and Standart 2010; Sharif and Conti 2013; Wu et al. 2014). Pat1 interacts with Lsm1-7 complex via Lsm2 and Lsm3 (Sharif and Conti 2013; Wu et al. 2014). The Pat1/Lsm1-7 complex is a well-characterized decapping activator that can bind to the 3’ end of the oligo-adenylated mRNA and promote mRNA decapping producing a 5’ monophosphorylated mRNA (He and Parker 2001; Chowdhury et al. 2007; Tharun 2009). Once decapped, the mRNA is rapidly degraded by the 5’ to 3’ exonuclease Xrn1. mRNA with a 5’ monophosphate are excellent substrates for Xrn1 and are quickly degraded (Muhlrad et al. 1994; 1995).

3.2. Exosome-mediated pathway

After deadenylation, mRNA can also undergo decay by exosome- mediated mRNA decay pathway (Figure 1). The cytoplasmic exosome complex along with several cofactors facilitates 3’-5’ mRNA decay pathway (Muhlrad et al. 1995). The exosome is a large complex with ten core subunits. Rrp41, Rrp42, Rrp43, Rrp45, Rrp46, Mtr3 forms a hexameric ring structure with which Rrp4, Rrp40 and Csl4 bind. These nine subunits together form the core ring structure that is suggested to act as a scaffold for proteins that target the exosome to RNA for degradation (Allmang et al. 1999b). The tenth subunit Rrp44 is the only 3’ to 5’ exonuclease and endonuclease of the complex (van

Hoof 2002; Lebreton et al. 2008; Schaeffer and van Hoof 2011). The exosome complex associates with the Ski complex, through Ski4 and Ski7, to carry out mRNA degradation in the cytoplasm. The Ski complex consists of Ski2, Ski3, and two Ski8 proteins. Ski2, an ATPase, is suggested to unwind the RNA that is entering the exosome complex and the association of Ski3 and Ski8 to Ski7 is required for 3’ to 5’ degradation of the mRNA (Araki 2001; Halbach et al. 2013). Once the mRNA is completely degraded from 3’ to 5’ degradation by exosome, Dcs1 hydrolyzes the released m7GDP cap to m7GMP. It is currently unclear how m7GMP is further processed (LIU et al. 2004).

4. Quality Control Mechanisms

Studies have shown that abnormal transcripts undergo very rapid decay, through many distinct pathways, resulting in a very low level of aberrant transcripts. Such faulty transcripts are generated due to 1) defects in mRNA biogenesis and processing, (Figure 2) 2) mutations in the gene or mutations incorporated it to the transcripts due to defective transcription (Figure 3).

4.1. Defects in mRNA biogenesis and processing 4.1.1. 5’ cap structure

During transcription, the m7G cap is added to the 5’ end of the mRNA. Capping occurs co-transcriptionally and is one of the earliest modifications on the mRNA (Rasmussen and Lis 1993). Cap structure is essential in processes like pre-mRNA splicing, polyadenylation,

nuclear export and translation (Krainer et al. 1984; Konarska et al. 1984; Shatkin 1985; Edery and Sonenberg 1985; Ohno et al. 1987; Hamm and Mattaj 1990; Izaurralde et al. 1992; Jarmolowski et al. 1994; Cooke and Alwine 1996). It also protects the mRNA from degradation by 5’ to 3’ exonucleases (Schwartz and Parker 2000; Wilusz et al. 2001a; Cougot et al. 2004).

Studies showed that mRNAs without 5’ cap were degraded 5’ to 3’ direction by Rat1 (Shimotohno et al. 1977; Kim et al. 2004). Rat1 is a paralog of Xrn1 that is present strictly in the nucleus. They are functionally redundant and can substitute for each other when forced to enter the other compartment (Johnson 1997). Mutations in the capping enzyme, Ceg1, or the cap methyltransferase, Abd1 have been shown cause a decrease in mRNA levels due to accelerated mRNA decay (Schwer et al. 1998; 2000; Cowling 2010).

4.1.2. Pre-mRNA splicing

Splicing is a process by which introns are removed from pre-mRNAs and the exons are ligated together. Defective splicing will lead to unspliced or mis-spliced transcripts that are eliminated by several degradation pathways. Mutations in genes related to splicing can also lead to the generation of aberrant transcripts. Unspliced or mis-spliced pre-mRNAs are either retained and degraded in the nucleus or exported to the cytoplasm for their decay. For example, in the mutant of a splicing factor, prp2-1, the splice-defective pre-mRNAs are retained in the nucleus and degraded by the exosome complex

(Plumpton et al. 1994; Bousquet-Antonelli et al. 2000). Pre-mRNAs that fail proper splicing were also shown to anchor to the perinuclear region of the nuclear pore complex, which probably prevents the nuclear export of these defective transcripts and might give them time for the exosomal degradation (Panse et al. 2002).

Pre-mRNAs that escape splicing or trapped as the lariat intermediates are proposed to be degraded by the nuclear pre-mRNA decay pathway (Bousquet-Antonelli et al. 2000). The nuclear Lsm2-8 complex, a complex similar to the Lsm1-7 complex in the cytoplasm, facilitates decapping of the pre-mRNA in the nucleus (Kufel et al. 2004). Decapped mRNAs are degraded by the nuclear 5’ to 3’ exonuclease, Rat1. Unlike the cytoplasmic decapping-dependent pathway of mRNA decay, the nuclear pre-mRNA decapping-dependent pathway is the minor pathway of degradation in the nucleus. The 3’ to 5’ degradation by exosome is the primary pathway of degradation in the nucleus (Bernstein and Toth 2012). Some unspliced pre-mRNAs may also be exported to the cytoplasm where they undergo degradation by NMD (Nonsense-Mediated mRNA Decay) (discussed below). As intron sequences contain an overrepresentation of translation termination signals, intron-containing pre-mRNAs in the cytoplasm will be targeted for decay by NMD (He et al. 1993; Sayani et al. 2008; Egecioglu and Chanfreau 2011).

Capping Pol II

m7Gppp

Capping enzyme complex 5’-3’ Degradation by Rat1

Splicing

Pol II Spliceosome m7Gppp Degradation by exosome Degradation by Nuclear mRNA decay Degradation by NMD

Polyadenylation Pol II

Clevage factors m7Gppp

AAAA m7Gppp PAP

Degradation by exosome

Exported to cytoplasm

Figure 2: Abnormal mRNA produced due to defects is mRNA biogenesis are eliminated.

4.1.3. 3’ end formation

When the transcriptional elongation complex reaches the polyadenylation signal, the pre-mRNA is cleaved, and poly(A) tail is added to the 3’ end of the mRNA. Like the cap structure at the 5’ end, poly(A) tail also plays a key role in the mRNA metabolism (Huch and Nissan 2014). Mutations in proteins required for polyadenylation, for example, pap1 mutants, lead to the production of mRNAs with no poly(A) tails that are rapidly degraded by nuclear exosome complex (Patel and Butler 1992; Mandart and Parker 1995). After polyadenylation, mRNAs are transported to the cytoplasm for translation. In mutants with defective mRNA export or THO complex (a complex required for coordination between transcription and mRNA processing), that couples mRNA export with transcription, accumulated mRNAs with longer poly(A) tails. It is believed that the failure to recycle the mRNP proteins to the nucleus is the cause for hyperadenylated mRNAs (Hilleren et al. 2001; Jensen et al. 2001; Libri et al. 2002). Studies showed that mRNAs with no or aberrant poly(A) tails are retained in the nucleus and degraded by the exosome complex (Hilleren et al. 2001; Jensen et al. 2001; Thomsen 2003).

4.2. Defects in mRNA due to genetic mutation 4.2.1. Nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) eliminates mRNAs with abnormal translation termination. NMD targets a wide variety of transcripts such as 1) mRNAs with premature termination codons

(PTC) that are introduced into the mRNA due to genomic mutations or transcriptional errors or mis-splicing events, 2) mRNAs that have long 3’ extended UTRs that alter the context of the stop codon to poly(A) tail, 3) mRNAs that contain alternative translational initiation sites that are out of frame with the original ORF, 4) mRNAs with upstream ORFs and 5) mRNAs with frameshifts (Losson and Lacroute 1979; Muhlrad and Parker 1999; Welch and Jacobson 1999; Das et al. 2000; Amrani et al. 2004; Gaba et al. 2005; Kebaara and Atkin 2009; Deliz-Aguirre et al. 2011).

m7Gppp AUG STOP Poly(A)

Nonsense-Mediated Nonstop decay Decay

m7Gppp AUG UAG STOP Poly(A) m7Gppp AUG STOP Poly(A)

Deadenylation independent 3’-5’ Exonucleolytic decay decapping and No-Go 5’-3’Exonucleolytic decay Decay:

m7Gppp AUG STOP Poly(A) Endonucleolytic Cleavage

Figure 3: Abnormal mRNA produced due to genetic mutation are eliminated by specialized degradation pathways.

Several studies indicate that translation of the PTC-containing mRNA is a key feature of NMD (Das and Das 2013). It is suggested that all mRNAs are monitored for PTCs during translation. When the

translation complex reaches a stop codon, the context of the stop codon is evaluated and determined if it is a PTC or normal stop codon. Currently, how a PTC is distinguished from a normal stop codon is unclear, but studies have shown that long distance between the stop codon and the poly(A) tail (which is short in normal mRNAs), also known a faux 3’ UTR, can trigger NMD (Amrani et al. 2004).

Although how a PTC is distigushed from a normal stop codon is currently under investigation, it is well established that both lead to translation termination by distict mechanisms. The current model is that binding of Upf1 to the long 3’ UTRs of the PTC containing transcript and the abnormal context of the stop codon to the poly(A) tail leads to inefficient translational termination. There after, Upf1 interacts with eRF3 to recruit other NMD factors for the degradation of the mRNA.

4.2.2. Non-stop decay

Non-stop decay or NSD is another quality control system that eliminates transcripts that lack proper termination codon (Frischmeyer 2002; van Hoof 2002). Translation of transcripts with premature polyadenylation or missense mutations in the stop codon or read through of the stop codon will cause the ribosome to reach the end of the transcript. When the ribosome reaches the 3’ end of the mRNA and is unable to terminate translation, it triggers NSD and recruits exosome and Ski complex. NSD is different from the regular 3’ to 5’ decay. Firstly, NSD requires GTPase activity of Ski7. Secondly, it

utilizes both endo and exonuclease function of Rrp44 of the exosome complex. The current model is that Ski7 recognizes the empty A-site and recruits Ski2-Ski3-Ski8 complex and exosome to degrade the mRNA from 3’ to 5’ direction (Schaeffer and van Hoof 2011).

4.2.3. No-go decay

No-go decay or NGD is triggered due to strong translational- elongation stalls on the mRNA by stem-loop structures (Doma and Parker 2006). The presence of rare codons, pseudoknot structures, frameshift sites, and poly-lysine or poly-arginine stretches also activate NGD (Doma and Parker 2006; Kuroha et al. 2009; Chen et al. 2010; Letzring et al. 2010; Belew et al. 2011). Dom34 and Hbs1, which are paralogs of the translational termination factors eRF3 and eRF1, the two components that promote NGD. The current model is that when elongating ribosome reaches a stem-loop structure, the ribosome stalls. Prolonged periods of empty ‘A’-site is recognized by Dom34, which then binds to it mimicking a charged tRNA leading to the release of the peptide-tRNA conjugate. It is believed that the Dom34 then recruits Hbs1 to the ribosome and together they might recruit the possible endonuclease that cleaves the mRNA. The endonuclease that cleaves the mRNA during NGD has not been identified yet. After the cleavage, the Xrn1 degrades the 3’ mRNA fragment, and the exosome complex degrades the 5’ fragment (Doma and Parker 2006).

5. Regulation of gene expression by mRNA decay factors

Several studies have shown that the processes of mRNA metabolism are linked to each other. Such coupling is suggested to enhance the synthesis of productive mRNA-protein complexes (mRNPs) and minimize the possibility of formation of defective transcripts, which would in turn facilitate the production of the desirable proteins (Hammell et al. 2002; Fasken and Corbett 2005; Houseley et al. 2006; Doma and Parker 2007; Parker 2012; Pérez-Ortín et al. 2013; Haimovich et al. 2013a; Huch and Nissan 2014; Radhakrishnan and Green 2016). Most mRNA degradation factors have been shown to regulate multiple steps in the process of eukaryotic gene expression (Table 1).

Table1. Cytoplasmic mRNA decay factors function in other processes of gene expression. Decay factor Function in mRNA decay Examples of other functions Reference Ccr4-Pop2-Not Components of the major Components of the CCR4-NOT (Denis 1984; Russell and Tollervey Complex cytoplasmic mRNA deadenylase transcriptional complex. 1992; Liu et al. 1998; Tucker et al. complex, involved in mRNA 2001; Liu et al. 2001; Tucker et al. poly(A) tail shortening. 2002; Thore et al. 2003; Alhusaini and Coller 2016) Pan2-Pan3 Subunits of the mRNA deadenylase Regulates the stoichiometry and (Boeck et al. 1996; Brown et al. Complex complex, controls poly(A) tail activity of post-replication repair 1996; Brown and Sachs 1998; length. complexes. Hammet et al. 2002; Wolf et al. 2014) Dcp1-Dcp2 Components of the decapping Forms cytoplasmic foci upon DNA (Beelman et al. 1996; LaGrandeur enzyme complex, removes the 5’ replication stress, can enter the nucleus and Parker 1998; Dunckley 1999; cap structure from mRNAs and and positively regulates transcription Vilela et al. 2000; Tkach et al. facilitate mRNA decay. initiation. 2012; Haimovich et al. 2013b) Pat1 mRNA-decapping activator, Represses translation, required for (Wang et al. 1996; 1999; Bonnerot required for protection of mRNA faithful chromosome transmission and et al. 2000; Noueiry et al. 2003; 3'-UTRs from trimming. maintenance of rDNA locus stability, Coller and Parker 2005; Hurto and associates with topoisomerase II, forms Hopper 2011; Ramachandran et al. cytoplasmic foci upon DNA replication 2011; Tkach et al. 2012) stress, phosphorylation by PKA inhibits P body foci formation, functions in tRNA retrograde transport.

Lsm1-7 complex Involved in degradation of Forms cytoplasmic foci upon DNA (Chowdhury and Tharun 2008; cytoplasmic mRNAs, binds to replication stress, involved in rRNA Herrero and Moreno 2011; Tkach et mRNA in the 3’ UTR. processing, can enter the nucleus and al. 2012; Wilusz and Wilusz 2013; positively regulates transcription Haimovich et al. 2013b) initiation. Dhh1 Stimulates mRNA decapping, Plays a role in translational repression (Bolsinger and Tanner 1993; Coller interacts with both the decapping and transcription, P body dynamics, et al. 2001; Bergkessel and Reese and deadenylase complexes. may have a role in mRNA export; 2004; Coller and Parker 2005; forms cytoplasmic foci upon DNA Carroll et al. 2011; Haimovich et replication stress, facilitates co- al. 2013b; a; Radhakrishnan et al. translational mRNA decay. 2016) Scd6 Promotes mRNA decapping Repressor of translation initiation, (Albrecht and Lengauer 2004; binds eIF4G through its RGG domain Rajyaguru et al. 2012; Tkach et al. and inhibits recruitment of the pre- 2012) initiation complex, may have a role in RNA processing, forms cytoplasmic foci upon DNA replication stress Stm1 Promotes mRNA degradation Protein required for optimal translation (Van Dyke et al. 2004; 2006; under nutrient stress, involved in TOR Balagopal and Parker 2008; Van signaling, protein abundance increases Dyke et al. 2009; Balagopal and in during to DNA replication stress, Parker 2011; Tkach et al. 2012) helps maintain telomere structure, serves as a ribosome preservation factor.

Edc1-Edc2 Directly activates mRNA Has a role in translation during heat (Dunckley et al. 2001; Schwartz et decapping, binds to mRNA stress, protein becomes more abundant al. 2003; Neef and Thiele 2009; substrate and enhances the activity and forms cytoplasmic foci in response Tkach et al. 2012) of decapping proteins Dcp1 and to DNA replication stress. Dcp2. Edc3 Plays a role in mRNA decapping, Localizes to cytoplasmic mRNA P (Kshirsagar and Parker 2004; affects the function of the bodies; forms cytoplasmic foci upon Tkach et al. 2012; He et al. 2014) decapping enzyme Dcp1p, and DNA replication stress. mediates decay of the Rps28 and Yra1. Xrn1 5'-3' exonuclease; involved in Component of cytoplasmic P bodies, (Larimer and Stevens 1990; mRNA decay. can enter the nucleus and positively Solinger et al. 1999; Geerlings et regulates transcription initiation and al. 2000; Sheth and Parker 2003; elongation, plays a role in rRNA Askree et al. 2004; Haimovich et maturation, required for microtubule- al. 2013b) mediated processes telomere maintenance; activated by the scavenger decapping enzyme Dcs1p. Exosome A ribonuclease complex with 3’-5’ Surveillance complex in the nucleus, (Allmang et al. 1999a; 2000; van processive exoribonuclease activity, removes abnormal pre-RNAs, Hoof 2002; Houseley et al. 2006; participates in a multitude of functions in pre-rRNA processing, Chlebowski et al. 2013; Lemay et cellular RNA processing and restricted to processing linear and al. 2014) degradation events. Degrades circular single-stranded RNAs defective mRNA from 3’-5’ (ssRNA) only. direction.

5.1. Modulation of Transcription by mRNA decay factors

As transcription is responsible for the synthesis of mRNA, early studies on the regulation of mRNA levels were concentrated on transcription. However, recent studies have shown that mRNA degradation can also play a critical role in the regulation of mRNA levels (Wang et al. 2002; Garneau et al. 2007; Molina-Navarro et al. 2008; Romero-Santacreu et al. 2009; Elkon et al. 2010; Castells-Roca et al. 2011). Several genome-wide studies have demonstrated that changes in transcriptional profiles are often accompanied by alterations in mRNA stability (Grigull et al. 2004; Molina-Navarro et al. 2008). For example, variations in the transcriptional activity during cell cycle can be correlated with alterations in mRNA stability (Talarek et al. 2010; Trcek et al. 2011). Several evidence indicate the existence of crosstalk between transcription and mRNA degradation. Rpb4 and Rpb7 heterodimer, subunits of RNA pol II, have been demonstrated to play a role in export, translation and mRNA degradation (Farago et al. 2003; Lotan et al. 2005; 2007; Goler-Baron et al. 2008; Harel-Sharvit et al. 2010). It has been suggested that Rpb4/7 heterodimer gets imprinted onto the mRNA during transcriptional elongation and serves as coordinator of transcription and mRNA decay to regulate the mRNA levels (Újvári and Luse 2005; Haimovich et al. 2013a).

Several studies have shown that factors involved in the process of mRNA degradation can also modulate transcription to regulate gene expression. The Ccr4-Pop2-Not complex is a well-characterized

deadenylase complex that is crucial for the general cytoplasmic mRNA degradation. Ccr4, the main deadenylase of the complex was initially identified as a transcriptional activator (Denis 1984; Draper et al. 1994; Tucker et al. 2001; 2002; Parker and Song 2004). Moreover, it was demonstrated that Ccr4-Not complex rescues the backtracked RNA pol II during transcription elongation (Kruk et al. 2011b; Miller and Reese 2012; Collart 2016). Although it has been shown that of Not proteins facilitate mRNA decay, they were initially identified as transcriptional repressors (Collart and Struhl 1994; Oberholzer and Collart 1998; Alhusaini and Coller 2016). Interestingly, the association of Ccr4-Not complex with elongating RNA pol II is facilitated by Rpb4/7, and Not5 of the Ccr4-Not complex promotes the export of Rbp4 into the cytoplasm (Villanyi et al. 2014; Babbarwal et al. 2014). As studies on the role of the Ccr4-Pop2-Not complex in transcription and mRNA decay were investigated independently, it is currently unclear how the Ccr4-Pop2-Not complex might facilitate the communication between transcription and mRNA decay.

Although decapping-dependent decay pathway is the major pathway of general cytoplasmic mRNA decay in yeast, deletion of the proteins involved in this pathway does not lead to dramatic increase in the total mRNA abundance (Haimovich et al. 2013b). Proteins involved in of cytoplasmic mRNA degradation, Dcp1, Dcp2, Lsm1, Dhh1, Pat1, and Xrn1, can also enter the nucleus (Teixeira and Parker 2007; Haimovich et al. 2013b; Muppavarapu et al. 2016; Huch et al. 2016). They were demonstrated to directly bind to the promoters, especially

to the promoters of the unstable mRNAs, and also to the other regions of the transcription unit. Some of the decay factors like Pat1 could facilitate transcriptional elongation when tethered to the promoters indicating that they might contain transcriptional activation domains (Haimovich et al. 2013b).

Although complete loss or mutations in the active site of Xrn1 has been shown have similar effects on mRNA decay, they had different effects on transcription suggesting that the effect of Xrn1 on transcription is independent of its effect on mRNA decay. Xrn1 was proposed to be the protein responsible for the buffering of total mRNA levels in yeast (Haimovich et al. 2013b; Braun and Young 2014).

5.2. Role of mRNA decay factors in Translation

The 5’ cap and the 3’ poly(A) tail of the mRNA plays a crucial role in both translation and mRNA degradation. These two structures promote translation initiation and inhibit degradation (Gallie 1991; Shatkin and Manley 2000). Several studies have established that mutations in the cap-binding complex inhibit translational initiation rates by increasing the deadenylation and decapping rates (Schwartz and Parker 1999). Decrease in translational initiation by mutations in the cap binding protein, eIF4E, leads to greater accessibility of the cap to decapping enzyme Dcp1/2 complex leading to an increase in the decapping rates. Furthermore, eIF4E can directly inhibit decapping in in-vitro assays (Schwartz and Parker 2000). These observations

suggest that the eIF4E and Dcp1/2 are in constant competition for the m7G cap and eIF4E needs to be exchanged to Dcp1/2 complex for decapping-dependent mRNA degradation. Similar to the cap structure, Pab1 bound 3’ poly(A) tail inhibits deadenylation by Ccr4-Pop2-Not complex and mRNA degradation (Wilusz et al. 2001b; Mangus et al. 2003).

Several transacting factors that negatively affect translation have been showed to enhance mRNA decapping and decay. For example, proteins like Pat1, Lsm1-7 complex, Scd6, Sbp1 and Dhh1 repress translation thereby promoting the mRNA decapping and general mRNA decay (Tharun et al. 2000; Coller and Parker 2005; Segal et al. 2006; Nissan et al. 2010; Carroll et al. 2011; Rajyaguru et al. 2012). Some mRNA decay factors like Pat1 and Dhh1 can recruits deadenylases to initiate deadenylation and then mRNA degradation (Coller et al. 2001; Ozgur et al. 2010; Cooke et al. 2010). On the other hand, RNA pol II subunits Rpb4/7 have been suggested to be imprinted onto the mRNA during transcription and regulate translational efficiency and mRNA decay of an individual mRNA or a class of mRNA in the cytoplasm (Choder 2011; Dori-Bachash et al. 2011; Haimovich et al. 2013a).

Recent studies have demonstrated evidences for cotranslational mRNA degradation and suggested that exchange of the translation initiation factors with decapping complex at the mRNA cap structure may occur while the mRNA is still associated with elongating ribosomes and optimality of the codons in the mRNA can determine

mRNA stability (Hu et al. 2009; 2010; Sweet et al. 2012; Presnyak et al. 2015). Ribosomes have been shown to elongate slowly on mRNAs containing non-optimal codons, and such mRNAs have been demonstrated to undergo decay in Dhh1 dependent manner (Radhakrishnan et al. 2016).

5.3. Ribosome Biogenesis and mRNA decay factors

Ribosomes are the ribonucleoprotein nanomachines that facilitates the conversion of genetic information in the mRNA into proteins. Ribosomes are composed of ribosomal RNA (rRNA) and ribosomal proteins (RPs). Biogenesis of ribosomes is one of the most complex and energy consuming processes of the cell. It is a multistep process involving, co-transcriptional processing, several chemical modifications, folding, and assembly of pre-rRNA, along with RPs into a massive ~3.3 MDa macromolecular machine.

Synthesis of the ribosome begins in the nucleolus; a non-membrane bound structure found in the nucleus. The nucleolus constitutes of chromosomal regions that contain approximately 100 to 200 copies of rDNA repeats from where the rRNA is transcribed. Ribosomla RNA is the most abundant RNA in the cell. A single 9.1 Kb rDNA repeat is transcribed to produce two primary transcripts namely, 35S pre-rRNA and 5S pre-rRNA. The 35S pre-rRNA contains the sequences of three mature rRNAs, 18S, 5.8S, and 25S rRNA that are parted by two internal transcribed spacers (ITSs) and flanked by two external transcribed spacers (ETSs). The 35S primary transcript undergoes a

series of cleavages and trimming steps to produce mature rRNAs. The 5S pre-rRNA is transcribed by RNA pol III and processed to mature 5S rRNA (Figure 4). Pre-rRNA processing can occur both co- transcriptionally and post-transcriptionally in yeast (Kos and Tollervey 2010; Woolford and Baserga 2013).

100-200 copies CEN TEL ChrXII L rDNA rDNA rDNA R

NTS1 NTS1 5’ETS ITS1 ITS2 3’ETS 5S 18S 5.8S 25S

5S pre-rRNA 35S pre-rRNA 18S 5.8S 25S 5S

20S pre-rRNA 27S pre-rRNA 18S 5.8S 25S

5S 18S 5.8S 25S

Figure 4: Pre-rRNA processing in S.cerevisiae. The pathway above shows a highly simplified version of pre-rRNA processing pathway.

The mRNAs of the 76 RPs (that are encoded by 137 genes) and ~ 200 trans-acting factors are transcribed by RNA pol II in the nucleus. They are then co-transcriptionally or post-transcriptionally modified, exported to the cytoplasm, translated and post-translationally modified into functional proteins. These proteins are then imported to the nucleolus to assemble with transcribing pre-rRNA into a huge pre- ribosome (Fatica and Tollervey 2002). Ribosome assembly can also occur co-transcriptionally in eukaryotes (Kos and Tollervey 2010).

The 35S pre-rRNA associates with RPs, trans-acting factors and many RNAs to form a 90S pre-ribosome, which undergoes sequential processing in the nucleolus, nucleus, and the pre-ribosomes are exported to the cytoplasm to form mature 60S and 40S ribosomal subunits. 25S, 5.8S, 5S rRNA, together with 46 large subunit RPs form 60S subunit and 18S rRNA together with 33 small subunit RPs, forms 40S subunit that fit together to form the complete 80S ribosome (Kressler et al. 1999; 2010).

The list of trans-acting factors involved in the biogenesis of ribosome is constantly growing. Many of these trans-acting factors are known to function in multiple other biological processes. For instance, Ded1 a translational initiation factor was shown to associate with both pre- 40S and pre-60S ribosomal subunits (De la Cruz et al. 1997; Schäfer et al. 2003).

Several mRNA degradation factors have also been implicated in ribosome biogenesis. Xrn1, a major cytoplasmic 5’-3’ exonuclease, is required for several processing steps in ribosome biogenesis (Stevens et al. 1991; Petfalski et al. 1998). Xrn1, together with Rat1 and another 5’-3’ exonuclease, Rrp17, was suggested to play a role in the trimming of 27SA3 pre-rRNA to 27SBs pre-rRNA and 26S pre-rRNA to 25S mature rRNA. Strains lacking Xrn1 or Rat1 or Rrp17 were shown to accumulate 5’ extended intermediates of 5.8S and 25S rRNA (Henry et al. 1994; Geerlings et al. 2000; Oeffinger et al. 2009).

The cytoplasmic Lsm1-7 complex that is required for general mRNA decay and the nuclear Lsm2-8 complex that promotes pre-mRNA degradation, have also been implicated in ribosome biogenesis. Depletion of components of these complexes led to abnormal rRNA processing and accumulation of pre-rRNA intermediates. Pre-rRNA intermediates co-immunoprecipitated with proteins of these complexes (Kufel 2003; Muppavarapu et al. 2016).

In our study (Paper I), we showed that the processing of pre-rRNA intermediates was delayed in the pat1 null mutant (see results and discussion of Paper I). We further show that the defects in the rRNA processing observed in the pat1∆ strain are probably due to dysregulation of the mRNA levels of genes related to ribosome biogenesis and ribosomal proteins (Muppavarapu et al. 2016).

5.4. P bodies and mRNA degradation

Eukaryotic processing bodies or P bodies are RNA-protein aggregates in the cytoplasm that have been proposed to control gene expression by regulating translation and mRNA degradation (Sheth and Parker 2003; Eulalio et al. 2007; Parker and Sheth 2007). As these dynamic structures contain mRNA degradation factors, they were initially thought to facilitate mRNA degradation (Sheth and Parker 2003). Although P bodies contain proteins involved in mRNA deadenylation, decapping-dependent mRNA degradation pathway, and NMD, components of cytoplasmic exosome-mediated mRNA degradation

pathway were not found in these granules indicating that they can probably modulate decapping-dependent mRNA degradation pathway to regulate gene expression (Sheth and Parker 2003; Cougot et al. 2004; Brengues 2005; Teixeira and Parker 2007; Buchan et al. 2010).

P bodies contain translationally repressed mRNAs, and their assembly is dependent on the pool of non-translating mRNA in the cell. Inhibition of translation initiation (which increases the non-translating mRNA pool) has been shown to promote P body formation and inhibition of translational elongation or trapping the mRNA in the polysomes (which reduces the free and non-translating mRNA pool) decreases P bodies (Teixeira et al. 2005). Moreover, P bodies are devoid of most translational initiation factors and ribosomes suggesting that P bodies most likely contain translationally repressed mRNAs (Ferraiuolo et al. 2005).

P bodies are proposed to assemble by a process in which smaller complexes, like Dcp1-Dcp2-Edc3 complex and Pat1-Xrn1-Lsm1-7 complex, along with mRNA aggregate to form larger RNA- protein complexes (Jain and Parker 2012). Edc3, Lsm4, and Pat1 are the proteins that are required for the assembly of P bodies in yeast. Although deletions of these proteins individually, has mild or no effect on P body formation, knockout of Edc3 together with Q/N rich C-terminus domain of Lsm4 (the mutant analyzed in Paper III) has been shown to completely eliminate P bodies. Deletion of Pat1 along with Edc3 has the strongest effect although it could be due to the inability of the Lsm1-7 complex to enter P bodies, as Pat1 is required

for the recruitment of Lsm1-7 complex to P bodies (Teixeira and Parker 2007; Decker et al. 2007; Buchan et al. 2008; Ozgur et al. 2010).

How P bodies affect mRNA decay is not yet clear. As inhibition of translational elongation does not affect deadenylation in yeast and as 3’-5’ components were not observed in P bodies, deadenylation and exosome-mediated mRNA degradation were thought to be not affected by P bodies (Beelman and Parker 1994; Sheth and Parker 2003; Brengues 2005; Hilgers et al. 2006). Although it is unclear how decapping is affected by P body formation, proteins that facilitate mRNA decapping are the core components of P bodies (Jain and Parker 2012).

We showed in Paper III that a strain that cannot form P bodies (edc3∆ lsm4∆C), displayed decresed mRNA stability. This decrease in mRNA stability is due to increased dependence on the deadenylation and decapping-dependent decay pathway. We speculated that P bodies, when induced, sequester mRNA deadenylation and decapping enzymes from the cytoplasmic mRNA pool and thus alter the decay mechanism of these mRNAs.

The following are the possible role of P bodies in the eukaryotic gene expression. First, they act as storage for the translationally repressed mRNAs. Second, they could protect the mRNA from degradation by sequestering away 5’- 3’ degradation pathway components from the cytoplasmic mRNA pool. Third, by sequestering the 5’- 3’

degradation pathway components, they promote 3’-5 exosome- mediated degradation and vice-versa.

Aims of the thesis

To investigate how mRNA decay factors regulate mRNA degradation to modulate gene expression.

Specific aims:

Paper I To understand the role of Pat1 in the nucleus.

Paper II To characterize the effect of edc3∆ lsm4∆C strain, a mutant that does not form visible P-bodies, on mRNA stability.

Paper III To understand the significance of the membrane association of the mRNA decay factors.

Results and discussion

Paper I

Pat1 is a multifunctional protein that has been shown to coordinate different cellular processes. It represses global translation and also activates decapping enzyme to promote mRNA degradation (Holmes et al. 2004; Coller and Parker 2005; Pilkington and Parker 2008; Nissan et al. 2010). As it interacts with many proteins that affect mRNA decay, it has been proposed to serve as a scaffolding protein to regulate the process of mRNA degradation (Nissan et al. 2010). Although Pat1 is predominantly cytoplasmic, where it is involved in major processes like translational repression and mRNA decay, several studies have provided evidence that Pat1 can enter the nucleus (Teixeira and Parker 2007; Marnef et al. 2011; Haimovich et al. 2013b). First, Pat1 has been shown to accumulate in the nucleus in the strain lacking Lsm1, a protein component of Lsm1-7/Pat1 complex and also a decay factor (Teixeira and Parker 2007). Second, the human homolog of yeast Pat1 (Pat1b) has been shown to shuttle between the nucleus and the cytoplasm (Marnef et al. 2011). Third, Pat1 was first identified as a topoisomerase II interacting protein, hence the name Pat1 (Protein Associated with Topoisomerase II) (Wang et al. 1996; 1999).

Although Pat1’s nuclear role is not yet clear, several studies have suggested many possible roles for Pat1 in the nucleus. Human Pat1b was demonstrated to be enriched in nuclear structures called splicing

speckles, which are irregularly sized dynamic structures that contain poly A+ mRNA and splicing factors. Therefore, it was suggested that Pat1 might interact with splicing factors and have a potential role in splicing-related functions (Marnef et al. 2011). Genome-wide transcriptomics studies and in vivo transcriptional assays established a role in transcription (Haimovich et al. 2013b). Pat1 has also been proposed to be essential for the tRNA retrograde transport, integrity of the centromeric chromatin and chromosome segregation (Hurto and Hopper 2011; Mishra et al. 2013).

We analyzed some of these potential roles and showed that Pat1 is not required for pre-mRNA splicing or nuclear pre-mRNA degradation, but it is necessary for normal rRNA processing and transcriptional elongation. Lack of Pat1 led to the accumulation of pre-rRNA processing intermediates, especially 35S, 27S and 20S pre-rRNA intermediates. Consistent with previous reports, deletion strains of Lsm1, Lsm6 and Lsm7 also displayed pre-rRNA processing defects (Kufel 2003; Li et al. 2009). The accumulation of the pre-rRNA processing intermediates was due to a delay in the pre-rRNA processing, as revealed by pulse-chase analysis.

Defective rRNA processing intermediates are known to be rapidly degraded by rRNA decay machinery, the nuclear exosome and the TRAMP complex (Allmang et al. 2000). To investigate if the lack of pat1 causes rRNA processing intermediates that are rapidly degraded by rRNA decay machinery and could not be detected in our northern blot and pulse-chase analysis, we analyzed double deletions strains.

Deletions of proteins involved rRNA decay machinery, nuclear exosome (Rrp6) and the TRAMP complex (Trf4) together with pat1, although did not reveal any new abnormal rRNA processing intermediates, it unveiled a novel synthetic sick relationship between Pat1 and the nuclear decay machinery.

Although we do not know the cause of the synthetic sick relationship, we speculate Pat1 might function in exosome and TRAMP related functions. First, as Pat1 together with Lsm1-7 complex can bind 3’ oligoadenylated mRNA in the cytoplasm, it potentially can bind TRAMP-mediated oligoadenylated RNAs that are targeted for decay, independently or together with Lsm2-8 complex, and facilitate the recruitment of the exosome for the degradation of these oligoadenylated RNAs (Chowdhury et al. 2007; 2014). Second, Pat1 can potentially bind TRAMP-mediated oligoadenylated RNAs for example, non-coding RNA, snoRNAs, etc. in the nucleus and might facilitate their processing (Grzechnik and Kufel 2008). Supporting this hypothesis, accumulation of snoRNAs was detected in pat1 deletion strain. Third, Pat1 might facilitate other TRAMP complex and exosome functions like determination of mRNA poly(A) length or control of pervasive transcription of noncoding RNA (Schmid et al. 2012; Tudek et al. 2014). Lastly, TRAMP complex protein Trf4 has a synthetically sick genetic relationship with topoisomerase TopI. This relationship is due to the accumulation of rRNA fragments generated in top1 mutant due to the formation of r-loops. As Pat1 is required for transcriptional elongation, it might have a similar relationship with TRAMP complex (Hage et al. 2010).

Although Pat1 can potentially function directly in rRNA processing similar to Lsm1 with which 20S pre-rRNA was co-precipitated (Kufel 2003), based on our results that Pat1 affects multiple steps and only delays rRNA processing, we theorized that lack of Pat1 might lead to down-regulation of transcription of ribosome biogenesis (RB) related genes as a class. To verify this hypothesis, we analyzed transcriptomics data from a study and found that the transcription of mRNAs related to RB was downregulated in the strain that lacks Pat1 leading to a decrease in the total abundance of these mRNAs (Sun et al. 2013).

Surprisingly, the results from the analysis of the transcriptomics data and our validation by Northern analyses did not correlate. Instead, the total abundance of most RB related mRNAs that we examined was increased in the pat1 deletion mutant. Interestingly, while mRNA levels of most RB related genes were upregulated in the pat1 deletion mutant, the ribosomal protein encoding mRNAs were not elevated suggesting that Pat1 affects the stability of RB related genes and not RP genes. Supporting this theory, mRNAs encoding RB related genes and RPs have been shown previously to have different decay profiles (Arribere et al. 2011). Moreover, the stability of RB related genes was demonstrated to be regulated by a motif on the mRNA that could be bound by Pat1/Lsm1-7 complex (Shalgi et al. 2005; Chowdhury and Tharun 2008). Taken together, these results suggest that Pat1 could potentially play a role in the decay of RB related genes as a class and the rRNA processing defects observed in pat1 deletion mutant are probably caused due to the dysregulation of mRNA levels of RB

related genes and RP genes.

In conclusion, several evidence suggest a model where Pat1, potentially together with Lsm1-7 complex regulates both mRNA degradation and transcription to facilitate the modulation of the levels of mRNA in the cell. Firstly, Pat1/ Lsm1-7 complex is a known decapping activator and mRNA degradation factor in the cytoplasm (Coller and Parker 2005; Nissan et al. 2010; Marnef and Standart 2010; Marnef et al. 2011; Haimovich et al. 2013b). Secondly, Pat1 and Lsm1 can nucleo-cytoplasmically shuttle. Accumulation of Pat1 in the nucleus in the deletion strain of Lsm1 suggests that Pat1 can enter the nucleus independent of Lsm1, but it requires Lsm1 to exit the nucleus. Alternatively, Lsm1 might be sequestering Pat1 in the cytoplasm via RNA granules called P bodies (Teixeira and Parker 2007). Thirdly, both Pat1 and Lsm1 have been shown to bind promoter regions (Haimovich et al. 2013b). Finally, Pat1 is required for transcriptional elongation in vivo assays, and it was shown to stimulate transcription upon recruitment to the promoter in another assay (Haimovich et al. 2013b; Muppavarapu et al. 2016). Finally, we demonstrate that the role of Pat1 in mRNA degradation and transcription are linked.

Paper II

Edc3 and Lsm4 are well-established components of the mRNA decay pathway. They have been demonstrated to be required for efficient mRNA decapping. Edc3 is also a decapping activator that can bind and directly stimulate Dcp2 (Harigaya et al. 2010; Nissan et al. 2010).

Similar to Pat1; Edc3 has also been proposed to play an imajor role as a scaffold for the assembly of the decapping complex (Decker et al. 2007; Nissan et al. 2010). Unlike other decay factors, lack of Edc3 does not affect genome-wide decay rates but instead it has been shown to specifically affect the stability of two mRNAs, RPS28B and YRA1 (Badis et al. 2004; Dong et al. 2007; Sun et al. 2013). Lsm4 is an essential protein component of the mostly cytoplasmic Lsm1-7 complex and the nuclear Lsm2-8 complex. Depletion of Lsm4 led to the accumulation of capped, deadenylated mRNAs, suggesting that it is required for mRNA decapping (Tharun et al. 2000). Deletion of Q/N rich C-terminus domain of Lsm4 was demonstrated to slightly affect mRNA stability (Decker et al. 2007; Reijns et al. 2008).

Edc3 together with Q/N rich C-terminus domain of Lsm4 plays a structural role in the formation of cytoplasmic RNA granules called P bodies. A strain lacking Edc3 and the Q/N rich C-terminal domain of Lsm4 (edc3∆ lsm4∆C) is defective in P body formation and lacks even the microscopically visible P bodies (Teixeira and Parker 2007; Decker et al. 2007). In this study, we characterized the effects of deletion of both Edc3 and Q/N rich C-terminus domain of Lsm4 on the mRNA metabolism in yeast. Our results show that, although the deletion of either Edc3 or the C-terminal domain of Lsm4 (edc3∆ and lsm4∆C strains) as expected, led to an increase in the stability of the mRNAs, deletion of both Edc3 and the C-terminal domain of Lsm4 (edc3∆ lsm4∆C strain), interestingly, resulted in the decrease in the stability of the mRNAs.

To further understand the mechanism of mRNA degradation in the

edc3∆ lsm4∆C mutant, genes of proteins in the mRNA decay pathways were mutated in the edc3∆ lsm4∆C background. Examination of these deletion mutants for their effect on mRNA half- lives revealed that the mRNA degradation in edc3∆ lsm4∆C mutant has a higher dependence on the deadenylation and decapping- dependent pathway and a concomitant reduction in the exosome- mediated pathway. To determine the source of the altered decay mechanism in edc3∆ lsm4∆C mutant we made following speculations.

First, increased dependence on the deadenylation and decapping- dependent pathway could be due to an increase in the enzymatic activities of the proteins involved in these steps. Evaluation of the activities of the deadenylation enzyme (Ccr4), decapping enzyme complex (Dcp1/2) or the exosome revealed that they are equally active in both wild-type and edc3∆ lsm4∆C mutant.

Second, the decreased stability of the mRNAs in edc3∆ lsm4∆C mutant could be the result of the altered levels of the mRNA decay factors. Consistent with our hypothesis, although the levels of Dcp2 were observed to be higher in edc3∆ lsm4∆C mutant compared to the wild- type strain, the levels of Dcp1 remained unaltered. As efficient Dcp2 activity depends on the Dcp1 levels (Beelman et al. 1996; Tharun et al. 2000; She et al. 2004; Floor et al. 2010), whose levels remained unaltered in edc3∆ lsm4∆C mutant compared to the wild- type strain when grown in glucose-rich medium, elevated levels of Dcp2 might not be the sole cause for the increased dependence on the deadenylation and decapping-dependent pathway. However, this possibility cannot be completely eliminated as the elevated levels of

Ccr4 (observed when the edc3∆ lsm4∆C strain was grown in galactose-rich media) have been demonstrated to increase the decay rate of PGK1, but not MFA2 (Tucker et al. 2002). Moreover, most of the additional Dcp2 in the edc3∆ lsm4∆C strain was found in the nucleus and might not be able to facilitate mRNA decay in the cytoplasm.

Third, the altered decay mechanism in edc3∆ lsm4∆C mutant could be due to rearrangement of RNPs on mRNAs, perhaps through altered decay protein levels or as a result of the lack of Edc3 and Q/N rich C terminus domain of Lsm4. Studies have shown that Edc3 and Pat1 can act as a scaffolding protein and can interact with Dhh1 separately, possibly to form different complexes to have distinct functions (Tharun et al. 2000; Badis et al. 2004; Harigaya et al. 2010; Nissan et al. 2010; Sharif et al. 2013). Lack of Edc3 in the edc3∆ lsm4∆C mutant might not be able to form certain complexes that are that are Edc3 specific. These observations together suggest a possible combinatorial code for mRNA decapping and degrdation (He and Jacobson, 2015).

Finally, the potential source for the reduced mRNA stability observed in the edc3∆ lsm4∆C mutant could be linked to its inability to function in the assembly of P bodies (Decker et al. 2007). P bodies may sequester the enzymes involved in deadenylation and decapping- dependent pathway and alter the mechanism of mRNA degradation. Hence, mRNAs not bound in P bodies have a different mechanism of decay and might decay more rapidly in their absence.

Paper III

As mRNA degradation factors and decay intermediates have been found to localize in P bodies, it was suggested that mRNA degradation occurs with in P bodies (Jain and Parker 2012). But a more recent study has demonstrated that mRNA decay can occur co- translationally on polysomes (Sweet et al. 2012). Other studies have indicated that mRNA decay factors can interact with membranes (Wilhelm et al. 2005; Kilchert et al. 2010). Such localized degradation can have important implications in gene expression. In this study, we showed that, mRNA decay factors are associated with membranes and their association with membranes is not dependent on stress, P body formation, translation and RNA.

Although the significance of the association between mRNA decay factors and membranes is not clear, we envision three non-mutually exclusive scenarios through which membrane association of mRNA decay factors can facilitate the regulation of gene expression. First, mRNA decay factors might be stored at the membranes until degradation is signaled. Second, by associating with membranes, mRNA decay factors might be sequestered away from the cytosol thus spatially separating the mRNA pool from mRNA decay factors. Third, the process of mRNA degradation might itself occur on membranes like the emerging concept in prokaryotes.

Conclusions

Paper I • Pat1 does not function in pre-mRNA splicing, nuclear pre- mRNA decay and nuclear snoRNA degradation. • Lack of Pat1 leads to delay in rRNA processing. • The rRNA processing defects observed in pat1 deletion mutant are due to improper mRNA degradation in the cytoplasm. • The absence of Pat1 leads to dysregulation of global mRNA levels that could result in decrease in transcriptional elongation as observed in pat1 deletion strain. • Pat1’s major role is in regulating mRNA stability.

Paper II • Edc3 and the glutamate/ asparagine- rich domain of Lsm4 are required for the formation of P bodies under glucose deficient or galactose conditions in Saccharomyces cerevisiae • Paradoxically, lack of both decapping activators Edc3 and the glutamate/ asparagine- rich domain of Lsm4, led to decrease in mRNA stability and increase in deadenylation. • This decrease in mRNA stability and increase in deadenylation in edc3∆ lsm4∆C strain is not due to the change in enzyme activity of mRNA decapping complex, deadenylation complex or exosome activity as they are similar to the wild-type strain in in vitro assays. • The abundance and localization of mRNA decay factors are altered in edc3∆ lsm4∆C mutant. • The decrease in mRNA stability is due to increased dependence on the decapping-dependent decay pathway.

• Lack Edc3 and the glutamate/ asparagine- rich domain of Lsm4 facilitate long-term survival of Saccharomyces cerevisiae.

Paper III • Proteins of mRNA decapping complex are components of larger structures as they sediment at lower centrifugal speed during differential centrifugation. • The association of the mRNA decay factors with the large structures is salt resistant and detergent sensitive suggesting that these large structures might be membranes. • Proteins involved in mRNA decay are membrane associated and float in a flotation assay. • mRNA degradation factors association with the membranes is independent of translation, stress and RNA.

Acknowledgements

“A journey is best measured in friends, rather than miles.” – Tim Cahill

I think a journey of life is best measured in memories and not in years. I have met so many wonderful people in the past five years of my PhD, without whom this thesis would not have been possible. I want to take this opportunity to thank all the people who helped me evolve not only as a scientist but also as a person.

First of all, I would like to express my heartfelt gratitude to you, Dr. Tracy Nissan, for giving me an opportunity to do PhD. Tracy, I have learnt a lot under your guidance, which would not have been possible otherwise. I am grateful to my PhD committee: Anders Byström (my co-supervisor), Vasili Hauryliuk, Jan Larsson, Stefan Björklund and Yuri Schwartz. Thank you so much for the valuable suggestions and feedbacks on my projects. Mona Byström - such a cheerful person you are! Thank you for your great company during our trip to Japan. Marcus Johansson - I am thankful for your positive criticism, encouragement, discussions, willingness to help, patience to teach tetrad picking and more. Glenn Björk - you are an inspiration to me! Thank you so much for the interesting discussions during our journal club meetings. Sara Wilson – Thanks a lot for being patient with me during the cell biology course. I admire how organized you are at work and it was a pleasure working with you!

Susanne - I would like to call you my second supervisor J. Thank you very much for teaching me a lot of things in lab. I am indebted to you for your time, effort and patience. I really appreciate the long talks, lunches, dinners and fikas we had! I would like to thank all the past and current members of Anders Byström’s lab and Marcus Johansson’s lab for the interesting scientific discussions during meetings and for all the fikas J. Especially, Hasan - you had saved my days so many times! From finding a chemical that I really needed, to fitting a bulb; you were always willing to help. Thank you very much for everything! Fu - thanks a lot for the YEPD plates and being so helpful and kind! Firoj - thank you very much for your help with the bioinformatics work and for nice talks. Yang - Can I share the water bath with you was my regular question J? Thank you Yang! Lifting up the huge rotor (on the -1 floor), help with yeast genetics and being an awesome guide

in Japan are only the few things (among many) that I would like to thank you for.

Gunilla - you are my awesome genie! I could ask you anything and you would have a solution for it J. Your cheerfulness had brightened many days of mine. Thank you so much for the interesting discussions and your kindness. Devendra Maurya – I thank you for patiently teaching me how to use the confocal microscope and for nice, little conversations. I am thankful to all the members of Vasili Hauryliuk’s group: Liis, Villu, Ievgen, Vallo, Yasuhiko - Thanks a lot for borrowing me things in the lab and for an awesome trip to the planetarium! My sincere thanks to my teaching colleagues, Sofie and Salah.

Geetanjali - Without you, I would not have been writing my thesis now Geet! I cannot thank you enough for being an amazing support system for me. I will always cherish those long talks, lunches and of course, your luscious cakes. I hope you have many more art exhibitions in the future and may be in the same place where I am living J. Anne-laure – thank you for all the interesting scientific discussions, fikas, lunches, parties and baking together! Roshani - Copenhagen is my best trip yet and I think it is because of the company I had ;) Thank you for being such an awesome you, roshani! Ala - it was a great pleasure to teach with you, Ala! I loved all the cute conversations, parties, get-togethers, lunches and dinners we had. Akbar, Lena, and Victoria –I truly had lots of fun partying, lunching, dining and dancing with you! Thank you so much! Sara-Thank you for your ID card J.

I am deeply grateful to many people who have made my stay in Umeå, a memorable one. In random order, I would like to thank-

Venki and bindu – for all the dinners you had cooked for me and for all the movies we watched together. I still think you are the best cook in Umeå, Venki J! Chaitanya – for your help in anything and everything. I am so glad I wrote you that mail. I hope you find good luck and happiness in life. Munender garu and sharvani – for being so kind, always inviting and willing to help. Karthik garu and Gowthami -for all the get-togethers and dinners. I wish you both all the very best! Kanaka – for long talks and hosting me during my trips to Uppsala. I know I can visit you anytime J. Shyam garu, Reshma, Ravi, Vidhya Ramya, Sujith, Madhavi garu,

Mohan garu, Lalitha, Rajesh garu, Harsha, Swarupa, Jaya, Rathi, and Pramod – for all your help, awesome dinners, amazing parties and delicious food!

I would also like to thank all the Mallu friends, Edvin, Maria, Lijo, Anjana, Sajna, Sinosh, Madhu, Nirmal, Deepu, Seema and Gireesh for a wonderful Onam sadya and a great party. Som, Indrayani - I will always treasure those memorable and joyful days we had, when you were in Umeå. Thank you! Special thanks to Chinmay, Chaitanya, Arun and Naresh! you guys are next in line J. I wish you good luck! Radha and Damini – Thank you for many warm conversations and all the amazing things we have done together. Svetlana and Shi Pey – I am extremely happy that I took the protein purification course and met you both. Thank you for the joyful time in Germany; shopping at Christmas markets, Christmas baking, lunches, dinners and many memorable moments.

Vijay – Arigato Daimas J! Ajeeth – you are the toughest person I know! Thank you for your encouragment to contemplate on my decisions. Surendra – I cannot thank you enough for the support you have been and for being such a big help! Sowmya – thank you very much for supporting me during my toughest times.

Anandi and Steffi – you have filled my heart with great joy and unforgettable memories. You are the best part of these five years J. Thank you for everything! I wish I had the fortune to meet you much earlier. I love you both! Hugs! MGR Rajan Uncle – Thank you for all the gifts and your help with the thesis J.

I express my deepest gratitude to my Family with out which I would not have been in Umeå. Dad and Mom- you are the source of my strength. Thank you for your support and for believing in me. Chandu and Sai - I love you!

Forever!

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