Most cellular proteins retain a stable concentration as cells grow and divide, but there are exceptions. Some cell cycle regulators change in concentration with cell size. In fission yeast, Cdc13 (cyclin B), an important activator of the core cell cycle kinase Cdc2 (CDK1), increases in concentration as cells grow. It has been proposed that the concentration of such cell cycle regulators serves as a proxy for cell size and makes cell cycle progression dependent on cell size, thereby contributing to cell size homeostasis. The underlying mechanisms for the size-dependent scaling of these cell cycle regulators are poorly understood. Here, I show that Cdc13 protein concentration, but not mRNA concentration, increases with cell size. Furthermore, only the nuclear, but not the cytoplasmic, fraction of Cdc13 increases in concentration as cell size increases. Computational modeling along with half-life measurements suggests that stabilization of Cdc13 in the nucleus plays an important role in establishing this pattern. Taken together, my results suggest that Cdc13 scales with time, and therefore only indirectly—not directly—with cell size. This leaves open the possibility that Cdc13 contributes to cell size homeostasis, but in a different way than originally proposed. / Master of Science / Cells maintain their size very efficiently, but how they manage to do so is not well characterized. It has been suggested that cells sense their size by the size-dependent concentration changes of cell cycle proteins. I have investigated how cyclin B may serve as such a proxy for cell size in fission yeast. My data suggest that fission yeast cyclin B indirectly scales with cell size through an unknown time-based mechanism.

Php4 is a subunit of the CCAAT-binding protein complex that has a negative regulatory function during iron deprivation in the fission yeast Schizosaccharomyces pombe. Under low iron conditions, Php4 fosters the repression of genes encoding iron using proteins. In contrast, under iron-replete conditions, Php4 is inactivated at both transcriptional and post-transcriptional levels. Our group has already described that Php4 is a nucleo-cytoplasmic shuttling protein, which accumulates into the nucleus during iron deficiency. On the contrary, Php4 is exported from the nucleus to the cytoplasm in response to iron abundance. Php4 possesses a leucine-rich NES (93LLEQLEML100) that is necessary for its nuclear export by the exportin Crm1. Our current study aims at understanding the mechanism by which Php4 is imported in the nucleus during iron starvation. Through microscopic analyses using different mutant strains, we showed that the nuclear localization of Php4 is independent of the other subunits of the CCAAT-binding core complex namely Php2, Php3 and Php5. Deletion mapping analysis of Php4 identifies two putative nuclear localization sequences (NLSs) in Php4 (171KRIR174 and 234KSVKRVR240). Using chimeric proteins that consist of GFP fused to Php4, we engineered substitutions of the basic amino acid residues 171AAIA174 and 234ASVAAAA240 and analyzed the functionality of both NLSs. We observed that both monopartite NLSs play critical role for Php4 nuclear localization. We also observed that mutant strains of cut15+, imp1+ or sal3+ exhibited defects in nuclear targeting of Php4, revealing that nuclear accumulation of Php4 is dependent on two karyopherin α (Imp1 and Cut15) and one karyopherin β (Sal3) receptors. Consistently, the Php4-mediated repression activity is abolished in the absence of two functional NLSs. Moreover, loss of Imp1, Cut15 or Sal3 resulted in increased expression of isa1+, which is a target gene of Php4. Co-immunoprecipitation assay (Co-IP) reveals physical interaction of Php4 with Imp1, Cut15 and Sal3 in vitro. Collectively, our results demonstrate that Php4 has two distinct NLS regions responsible for its nuclear localization. Furthermore, karyopherin α and β receptors play a role in the nuclear import of Php4. Because Php4 is essential for growth under low iron conditions, the presence of two NLSs would ensure the protein to reach its nuclear destination when cells undergo a transition from iron-sufficient to iron-limiting conditions.

The interval between meiotic nuclear divisions can be regarded as a modified mitotic cell cycle where DNA replication is blocked. Mechanisms regulating this critical aspect of meiosis that allows haploid cells to be generated from a diploid progenitor were investigated in this project. Licensing is restricted after meiosis I due to down-regulation of Cdc18 and Cdt1. Late meiotic expression of Cdc18 and Cdt1, which load the MCM helicase onto replication origins, can lead to partial DNA replication after meiosis I. This implies that block to initiation via licensing forms an important component of this regulation. As detecting any minor DNA re-replication after meiosis I requires a technique more sensitive than flow cytometry for detection of total cell DNA contents, I also investigated a procedure to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast. Additional inactivation of Spd1 or stabilization of Dfp1 after MI when Cdc18 and Cdt1 are also expressed does not enhance re-replication, but cyclin-dependent kinase Cdc2 plays a role in preventing re-replication during the MI-MII interval. Unexpectedly, when the licensing block is subverted, replication forks only move a short distance in the interval between meiosis I and II, implying that the elongation step of DNA replication is also inefficient. In addition, I show that the regulation of entry into meiosis II is not delayed by a partial round of DNA replication or DNA damage, indicating that replication and DNA damage checkpoints do not operate in late meiosis.

Thesis advisor: Charles S. Hoffman / In this thesis, I describe the development of a screening platform for detecting PDE8A inhibitors using the cAMP-dependent glucose sensing pathway of the fission yeast Schizosaccharomyces pombe, which led us to discover several PDE8A selective inhibitors. In this system, the only PDE of the fission yeast is replaced with mammalian PDE8A1 in strains that have been engineered such that PDE inhibition is required to allow cell growth. Using this system, I screened 56 compounds obtained from PDE4 and PDE7 high throughput screens (HTSs) and identified a PDE4-PDE8 dual specificity inhibitor. Using this as a positive control, I developed a robust high-throughput screen (HTS) for PDE8A inhibitors and screened 240,267 compounds at the Harvard Medical School ICCB Screening Facility. Approximately 0.2 % of the screened compounds were potential PDE8A inhibitors with 0.03% displaying significant potency. Secondary assays of 367 of the most effective compounds against strains expressing PDE8A (both full length and catalytic domain), PDE4A and PDE7A or PDE7B led to the selection of structurally diverse compounds for further testing. To profile the selectivity of twenty-eight of these compounds, dose response assays were conducted using 16 yeast strains that express different PDE isoforms (representing all PDE families with the exception of the PDE6 family). These assays identified compounds with different patterns of inhibition, including structurally-distinct PDE8A-specific inhibitors. By evaluating the effects of these compounds for steroid production in mouse Leydig cells, biologically active compounds that can elevate steroid production were identified. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Cell polarisation is a key biological process crucial for the functioning of essentially all cells. Regulation of cell polarity is achieved through various processes determined by both internal and external factors. An example of the latter is that cell polarity can be disrupted or lost as a consequence of a variety of external stresses. When facing such stresses, cells adapt to unfavourable conditions by activating a range of molecular signalling pathways, collectively termed ‘stress response’. Despite the connections between external stress and cell polarity, whether stress-response signalling regulates cell polarisation and what the molecular basis for such regulation remains an open question. The fission yeast Schizosaccharomyces pombe presents an excellent biological platform to study the complexity of cell polarity regulation on a systematic level. This study is aimed at understanding the functional relationship between stress-response signalling and maintenance of cell polarity in this model organism. The findings presented in this thesis set the basis for establishing a functional link between the activation of the S.pombe stress-response pathway and the activity of the master regulator of cell polarity- the Rho GTPase Cdc42. Here, I describe experiments that identify an active involvement of the stress-response mitogen-activated kinase (MAPK) Sty1 in the dispersal of active Cdc42 from the sites of growth. This new role for Sty1 occurs independently from its involvement in transcription regulation and other previously identified signalling pathways involving Sty1. Furthermore, I also find that Sty1’s involvement in Cdc42 regulation has direct implications for fission yeast physiology as it is essential for the maintenance of cellular quiescence upon nitrogen starvation. This thesis also focuses on identifying the targets of Sty1 orchestrating the active Cdc42 disruption. Here, I describe a candidate-based approach, where I investigate the role of proteins from the Cdc42 regulatory network during Sty1 activation. Additionally, I present a global phospho-proteomics approach to identify novel targets of Sty1 and offer preliminary findings which might explain Sty1’s involvement in Cdc42 regulation.

Eukaryotic centromeres are the region upon which kinetochores assemble, directing attachment of spindle microtubules and faithful segregation of chromosomes during mitosis and meiosis. Except for a transient disruption in mitosis when chromosomes are segregated, centromeres of fission yeast Schizosaccharomyces pombe remain closely associated with the nuclear periphery. Similar to multicellular eukaryotic centromeres, they also maintain unique chromatin architecture, with a central core defined by the presence of the conserved centromeric histone H3 variant CENP-A, designated Cnp1 in S. pombe, that is flanked by histone H3 containing heterochromatin. While much progress has been made in understanding chromatin-associated factors important for proper centromere function, many questions remain. In order to gain a better understanding of the factors involved in centromeric chromatin structure, we affinity purified and defined by mass spectrometry interactions among select proteins that had been implicated in proper Cnp1 localization and centromere function. These biochemical purifications revealed several proteins that may be involved in Cnp1 localization. Purification and analyses of Cnp1 also led us to the identification of the Mlp1/Tpr nucleoporin homolog Nup211. We have found that Nup211 interacts with components of the inner nuclear basket of the nuclear pore, and co-purifies with centromeric chromatin proteins. Cells lacking Nup211 have substantial chromosome segregation defects, as observed by synthetic growth assay, flow cytometric analysis, and fluorescent microscopy. A series of immunoprecipitation experiments have revealed that Nup211 associates with centromeric DNA, and that, surprisingly, cells lacking Nup211 have increased histone H3 lysine 9 methylation, a marker of heterochromatin, and a reduction in Cnp1 levels at the central core. Moreover, cells lacking Nup211 have decreased transcription at centromeric loci, disruption of the stereotypical nucleosome structure found at the central core of S. pombe, and show striking changes in the distribution of heterochromatic foci in the nucleus. By demonstrating that Nup211 is essential for the maintenance of normal central core chromatin state, these studies have shed light on a novel role for Nup211 in proper centromere structure and function in S. pombe, and suggest that Nup211 may play a role in preventing the invasion of flanking pericentric heterochromatin into the central core of centromeres.

Dsk1 is the Schizosaccharomyces pombe functional homolog of human SRPK1, an SR protein kinase that regulates localization and function of SR protein splicing factors involved in transcription, alternative splicing, and mRNA export. It has been shown that a Dsk1 deletion strain of S. pombe is sensitive to exposure to butylated hydroxyanisole (BHA), a phenol derivative commonly used as a food preservative. Little is known about how BHA interacts with cells on a functional level, although it has been shown to be cytotoxic and tumorigenic. The aims of this thesis are to study the effect of BHA on eukaryotic cells and the possible involvement of Dsk1 protein kinase in the cellular response network to BHA through the use of fluorescence microscopy. The results showed that in BHA-treated cells, Dsk1 exhibits reduced nuclear localization and increased incidence of cytoplasmic clusters as well as a series of changes in cellular morphology. These observations imply that the function of Dsk1 is altered in response to BHA, consistent with genomic data collected by the Tang Lab. Thus, this study provides a basis for a series of future studies that will reveal in more detail how BHA affects fission yeast cells, and potentially gene or protein functional homologs in human cells.

Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side. Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules. By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.

Exonic sequences of eukaryotic genes are interspersed with introns which when accurately removed from the primary transcript (pre-mRNA) results in a functional transcript. These splicing reactions are carried out by the spliceosome, consisting of U1, U2, U4, U5, U6 snRNAs and 150 non-snRNP proteins, which assemble onto the pre-mRNA and catalyzes the two invariant transesterification reactions (Will and Luhrmann, 2006). The flexibility in choice of splice sites allows for alternative splicing which has immensely contributed to eukaryotic genome evolution and in diversifying the metazoan proteome (Nilesen and Graveley, 2010). Dynamic yet ordered interactions between U2, U5 and U6 snRNAs and Prp8, Prp16, Prp17, Prp18, Slu7 and Prp22 splicing factors are required in vitro for second-step of splicing of budding yeast and human model transcripts (Umen and Guthrie, 1995a; Horowitz, 2012). ScSlu7 aids 3’ss selection while its strongly associated partner ScPrp18 stabilises U5 snRNA-exonic interactions (James et al., 2002; Aronova et al., 2007). These factors are dispensable in vitro, for the splicing of introns with short branch nucleotide to 3’ss distances (Brys and Schwer, 1996; Zhang and Schwer, 1997). Nearly 43% of fission yeast genes have short introns, with degenerate splice-signals and unconventional Py(n) tracts (Kuhn and Kaufer, 2003). As these features differ extensively from budding yeast and are interestingly more representative of fungal and other eukaryotic introns, fission yeast is an attractive unicellular model to investigate alternate splice-site recognition and assembly mechanisms. Mechanistic details of the second catalytic step are poorly understood in fission yeast. Strikingly, mutations in 3’ss and Py(n) tract intronic cis elements, known to block second step splicing in budding yeast, cause pre-catalytic arrest with unspliced pre-mRNA accumulation in fission yeast (Romfo and Wise, 1997). Studies in our laboratory focussed on understanding the functions for fission yeast SpPrp18 and SpSlu7 predicted to be second-step factors, revealed remarkable differences as compared to their budding yeast counterparts. Unexpectedly, SpPrp18 and SpSlu7 were found by our lab to be required before catalysis and these proteins do not directly associate with each other. Genome-wide splicing studies in a missense slu7-2 mutant indicated widespread yet intron-specific splicing functions for SpSlu7 (Banerjee et al., 2013). Crucial functions were attributed to helix-5 and conserved region loop of SpPrp18 and in vivo splicing analysis in selected cellular transcripts in a missense mutant (V194R) also revealed intron-specific functions (Thesis, N Vijaykrishna). In this study, we have advanced our understanding of SpPrp18 functions by identifying its global substrates and correlating with its intron-specific roles. Through molecular and genetic approaches, we have probed its role in splicing/spliceosome assembly. We identified intronic features within substrates that increase the propensity for the requirement of SpSlu7 for efficient splicing. Further, using findings from the genome-wide alternative splicing patterns in SpSlu7 and SpPrp18 mutants, we have attempted to understand their role in splice-site choice and thus alternative splicing. Ia. Understanding global splicing functions and spliceosomal interactions of fission yeast splicing factor SpPrp18 Since SpPrp18 is an essential gene, our lab generated the strains (prp18-5int [V194R] and WTint), where the thiamine-repressible promoter allowed conditional expression of wild-type or mutant allele integrated at the heterologous leu1 locus. Splicing efficiency of certain cellular transcripts with differing intron characteristics was assessed by semi-quantitative RT-PCR studies and the data suggested intron-specific SpPrp18 roles (in collaboration with Vijaykrishna N). This prompted us to investigate the global splicing role for SpPrp18 for which we used splicing-sensitive microarrays having custom-designed probes to distinguish unspliced pre-mRNA and spliced mRNA for every individual pombe intron. RNA from prp18-5int (V194R) and WTint cells was used in these experiments. We derived a stringent dataset of 258 introns which were statistically significant and correlated in two biological replicate RNA samples, for various probes. Hierarchical clustering of this dataset showed that the depletion of wild-type SpPrp18 triggered a range of splicing phenotypes like (A) pre-mRNA accumulation with mRNA reduction (B) pre-mRNA accumulation (C) spliced mRNA reduction and (D) unchanged pre-mRNA and mRNA levels. Statistical analysis of cis motifs that may correlate with the substrate-specific SpPrp18 splicing functions was done, but the data showed a lack of a global discriminatory primary sequence feature. However, a subtle intron-specific role for Py(n) tracts located between 5’ss and BrP was deduced for SpPrp18. This lead was validated by examining the in vivo splicing efficiency of minitranscripts with wild-type or an altered Py tract length, carried out for a SpPrp18 dependent and an independent intron. To specifically address if SpPrp18 activity was required for second-step splicing we investigated, using primer extension analyses, for lariat intron-3’exon species, an intermediate formed after step 1. We observed that even in prp18-5int dbr1∆ double mutants (where lariat molecules are not degraded) the cells accumulate only unspliced pre-mRNA and not lariat intermediates, a signature of an early arrest prior to the first transesterification reaction. Strengthening these findings, positive genetic interactions were noted between prp18-5int and ts mutants in two factors (U2AF59 and SpPrp1) involved in precatalytic spliceosome assembly and activation. On the whole, our genome-wide studies indicate intron-specific pre-catalytic functions for SpPrp18 supported by genetic interactions with early acting splicing factors involved in spliceosomal assembly and activation. Ib. Identification of intronic features that determine substrate-specific splicing functions for SpSlu7 In vitro studies with ScSlu7 and hSlu7 show their influence in 3’ss selection when BrP to 3’ss distance is greater than 7 nts and 23 nts respectively; but the global substrates are not known in either species (Brys and Schwer, 1996; Chua and Reed, 1999b). Genome-wide analysis of the splicing efficiency changes in cells with the mis-sense spslu7+ mutant (slu7-2), previously carried out in our lab, revealed a spectrum of splicing defects (Banerjee et al., 2013). To further understand the intron context-specific roles for SpSlu7, we examined intronic cis features that may correlate with SpSlu7 dependence. Statistical analyses of the affected (422 introns) and unaffected categories (90 introns) revealed that intron length, BrP to 3’ss distance and AU content are multiple discriminatory cis features that govern SpSlu7 splicing functions. To assess the contribution of these intronic features we tested whether altering these cis elements changes a transcript’s dependency (or otherwise) on SpSlu7 by RT-PCR analyses. For these studies, we generated plasmid expressed mini-genes containing the respective wild-type intron or intron with altered BrP-3’ss distances. We used nab2+ I2 as a case of an intron spliced independent of SpSlu7 and rhb1+ I1 as a representative for SpSlu7 dependent intron. Experiments testing their in vivo splicing status proved that BrP-3’ss distance is a cis feature that dictates SpSlu7 splicing functions in a context-dependent manner. The intronic AU content particularly between the 5’ss and the BrP was assessed in minigene constructs where a chimeric intron was generated by swapping the low AU containing sequences in the 5’ss to BrP stretch of cdc2+ I2 with AU rich bpb1+ I1 5’ end sequences. The results reaffirmed that low intronic AU content particularly at the 5’ end co-relates with SpSlu7 dependency. Hence, we have deduced novel intronic elements, which perhaps in combination, create a contextual dependence for SpSlu7 to facilitate efficient splicing. II. Alternative splice-site selection in fission yeast and studies on the role of splicing factors SpSlu7 and SpPrp18 Budding yeast second-step splicing factors ScSlu7 and ScPrp18 mediate 3’ss choice in the single intron containing transcripts. Fission yeast genome encodes cis and trans factors that promote alternative splicing similar to higher eukaryotes. In this study, we have devised a data analysis pipeline to identify alternative splice events in multi-intronic transcripts of fission yeast. Further, we utilised this information to interrogate the global role for SpSlu7 and SpPrp18 in alternate splice site selection. We mapped the microarray probe sequences corresponding to all theoretically possible non-consecutive splice junctions of S. pombe transcripts onto two independent experimental next-generation (NGS) transcriptomes from wild-type samples and identified 104 exon skipping events with NGS reads more than 3 (Wilhelm et al., 2008; Rhind et al., 2011). We further generated a stringent list of ten exon skipping events having high sequence reads as well as raw intensity value in our microarray experiments with wild-type cells. Two representative events from this list, an abundant rps13+ exon 2 skipped alternative mRNA and less abundant ats1+ exon 3 skipped alternative mRNA were then taken up for experimental analyses by semi-quantitative RT-PCR assays. We confirmed these events and further noted that SpSlu7 and SpPrp18 were required for the constitutive splicing of ats1+ E2-I2-E3-I3-E4 cassette. On the other hand, SpSlu7, and not SpPrp18, exerted a subtle influence on the skipping of exon 3. In addition to exon 3 skipped mRNA, we detected an intron 3 retained ats1+ alternative mRNA (E2-E3-I3-E4) in wild-type cells. Assessment of this event in cells metabolically depleted of SpSlu7 and SpPrp18 showed a reduced abundance of this species in both instances. This suggests a role for functional SpSlu7 and SpPrp18 in retaining intron 3 in ats1+ transcripts in vivo. Among the ten microarray probes, custom-designed to detect specifically the mRNA isoforms arising from altered use of donor 5’ splice sites, we were able to detect in wild-type cells the utilisation of a downstream alternate 5’ss in intron 1 of D-Tyr-tRNA deacylase. Comparative assessment of this splicing event in prp18-5int and slu7-2 mutant cells revealed that SpPrp18 is preferentially required for the utilisation of its alternative 5’ss and such a role has not yet been attributed to its budding yeast and human homologs. On the other hand, SpSlu7 was required equally for utilisation of canonical and non-canonical 5’ss. Differential requirement for SpSlu7 for the utilisation of an upstream non-canonical 3’ss and the canonical 3’ss in DUF3074 intron 1, was noted. This role of SpSlu7 in 3’ss selection is similar to that known from in vitro studies of its budding yeast and human counterparts. Overall, we identified and experimentally validated novel alternate splice events in fission yeast and we infer an important role for SpSlu7 and SpPrp18 in both 5’ss and 3’ss selection.

Epigenetics is an area of biology that studies heritable changes in gene ex- pression without any change in the DNA sequence. The most studied epige- netic mechanisms are DNA methylation, RNA interference and histone mod- ifications. There are over 130 different modifications that can be attached to histones, and the most commonly studied are methylation, acetylation, phos- phorylation, sumoylation and ubiquitination. The modifications, spread out through the genome, form the histone code, which recruits transcription fac- tors and modifies the accessibility of the DNA, which results in either active or silenced transcription. The silenced form of chromatin is known as heter- ochromatin and is usually found in regions of the chromosome that need to be highly regulated. To study epigenetics, the model organism Schizosac- charomyces pombe is used widely used. S. pombe is a single cell, rod shaped, fission yeast. The simplicity of S. pombe and its similarities to high- er eukaryotes makes it a good model organism for studying epigenetics. We find that, when mutating evolutionary conserved amino acids in the Clr2 protein, which is involved in heterochromatin formation in S. pombe, there is a change in silencing in different heterochromatic regions. When constructs of Clr2 with the BAH domain deleted are overexpressed, there is an increase in silencing in the central core centromere of chromosome II of S. pombe.