Tesi sul tema "Fission pathways"

At the end of G1 phase, cells have to decide between continue proliferation or remain in a quiescent state (G0). This decision point, known as “Start” in yeast and “Restriction Point” in metazoans, marks irreversibly the commitment to the completion of a new cell cycle, and is regulated mainly by the activity of the G1 CDK and the induction of the G1-to-S transcriptional program. The MBF transcription factor complex (functional homolog of pRB-E2F in metazoans) drives the G1-to-S transcriptional wave in the fission yeast Schizosaccharomyces pombe. We have previously described how the co-repressors Nrm1 and Yox1 bind to MBF complex at the end of S phase, inhibiting the MBF activity. However, the mechanisms involved in the activation of MBF at the onset of an unperturbed cell cycle have remained elusive. Here, we show that Nrm1 is the responsible for the activation of the MBF-dependent transcription through a two-step mechanism. Its phosphorylation by CDK1 and its posterior degradation by APCSte9 induce the irreversible MBF activation until the end of S phase. We have also studied the role of chromatin remodelers in the control of the G1-to-S transcriptional program. In this sense, we have found that chromatin-remodeling complexes SWI/SNF and RSC are recruited to MBF-regulated genes, having a clear impact in the activation of the G1-to-S transcriptional wave. Furthermore, we have created a short-lived fluorescent reporter to measure small and transient changes in the MBF activity in vivo by flow cytometry, to further identify new MBF regulators.
Al final de la fase G1, les cèl·lules han de decidir entre continuar proliferant o romandre en un estat de quiescència (G0). Aquest punt de decisió, conegut com “Start” en llevats o “Restriction Point” en metazous, compromet irreversiblement a les cèl·lules a completar el següent cicle cel·lular, i està principalment regulat per l’activitat CDK de G1 i per la inducció del programa transcripcional de G1/S. El complex MBF (homòleg funcional de pRB-E2F en metazous) es el factor de transcripció encarregat de la inducció de l’onada transcripcional de G1/S en el llevat de fissió Schizosaccharomyces pombe. Anteriorment, vam descriure com els repressors Nrm1 i Yox1 s’uneixen al complex MBF al final de la fase S per inhibir la seva activitat. Fins ara, els mecanismes implicats en l’activació de MBF a l’inici d’un cicle cel·lular no pertorbat s’han mantingut desconeguts. En aquest treball, hem vist que Nrm1 es el responsable de l’activació transcripcional depenent de MBF mitjançant un mecanisme de dos passos. La seva fosforilació per CDK1 i la seva posterior degradació per APCSte9 donen lloc a l’activació irreversible de MBF fins al final de la fase S. També hem estudiant el paper dels remodeladors de cromatina en el control del programa transcripcional de G1/S. En aquest sentit, hem trobat que els complexes remodeladors de la cromatina SWI/SNF i RSC són reclutats als gens regulats per MBF i tenen un clar impacte en l’activació transcripcional de G1/S. A més, hem creat un reporter fluorescent de vida curta per mesurar canvis petits i transitoris de l’activitat MBF in vivo mitjançant citometria de flux, per a poder identificar nous reguladors de MBF.

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.

La quiescence et la prolifération reflètent deux stades cellulaires fondamentalement différents, mais il existe très peu d'informations sur la manière dont les cellules maintiennent la stabilité de leur génome en quiescence. En utilisant des levures de fission privées d’azote comme modèle de quiescence, notre laboratoire a démontré que les cellules subissent non seulement les dommages de l’ADN aux indépendantes de la réplication linéairement avec le temps. Dans les travaux en cours, nous avons démontré que les mutations accumulées au cours de la phase d'arrêt de la croissance subissent un processus de sélection en quiescence similaire à celui observé chez E. coli. La sélection favorise les mutations qui affectent les fonctions des gènes des 3 voies de la MAP-kinase (mkh1, pek1, pmk1) et de la SAP-kinase (win1, wis1, sty1) et de leurs cibles en aval (pmc1, sgf73, tif452). Ces gènes sont impliqués dans la signalisation cellulaire centrale qui régule la prolifération, la différenciation et la mort cellulaire conservée chez toutes les espèces eucaryotes, de la levure à l'homme. Des mutations dans des composants de la voie S/MAPK ou dans ses régulateurs sont associées à de multiples maladies chez l'homme, dont certains cancers et une mort neuronale dégénérative en fonction de l'âge. Les cellules libèrent des traces d'azote lors de leur mort, ce qui déclenche l'entrée dans le cycle cellulaire des cellules encore en vie. Les cellules sauvages ne peuvent compléter un cycle et meurent, libérant davantage d'azote. Les mutants de la voie S/MAPK sont caractérisés par une capacité d'entrée dans le cycle différente en fonction de la concentration d'azote disponible ce qui entraine une résistance à la mort cellulaire
Quiescence and proliferation reflect two fundamentally different cellular stages, yet very limited information exists on how cells maintain their genome stability in quiescence. Using nitrogen-starved fission yeast as a model for quiescence, our laboratory has demonstrated that cells are not only subject to DNA damage in G0 but also accumulate replication-independent mutations linearly with time. In our current work, we have demonstrated that mutations accumulating in growth-arrested phase undergo a selection process in quiescence similar to that observed in E. coli. Selection favors mutations that affect functions of the genes of the MAP-kinase (mkh1, pek1, pmk1) and SAP-kinase pathways (win1, wis1, sty1), and their downstream targets (pmc1, sgf73, tif452). These genes represent core cellular signaling that regulates cell proliferation, cell differentiation, and cell death conserved among all eukaryotic species from yeast to human. Mutations in components of the S/MAPK pathways and their regulators are associated with multiple diseases in humans, primary cancer and degenerative neuronal death accumulated with ageing. In this work, we have demonstrated that wild-type cells dying in quiescence release traces of nitrogen that triggers the viable population to exit from quiescence. The wild-type cells are dying during their entry into S-phase releasing more nitrogen. Thus, mutants in the S/MAPK pathways are better scavengers and selection in quiescence is characterized by the ability of the mutant to resume cycling in quiescence coupled with a resistance to programed cell death

Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is involved in both oxidative stress and signaling cascades in a dosage dependent manner. Its toxicity is partially explained through reactivity with iron via the Fenton reaction. Iron, indispensible for many cellular processes, is thus tightly regulated to balance between need and toxicity. Using fission yeast as a model system, we explored the relationship between H2O2 and the iron starvation response system, specifically whether cross-talk allowed mutual regulation that could prevent synergistic toxicity of ROS via diminishing iron quantity. We screened around of 2700 haploid Schizosaccharomyces pombe deletion mutants in different oxidative stress agents, identifying new genes amongst which fep1, pcl1 and sib2 are involved in iron homeostasis. H2O2, unexpectedly, triggers transcriptional iron starvation response, including enhanced iron import and decreased iron consumption. Over-expression of several antioxidant proteins, in particular heme-containing catalase, causes strong iron consumption within the cell, triggering the iron starvation pathway accidentally. Furthermore, glutaredoxin Grx4 contains an iron-sulfur cluster (ISC) involved in iron sensing, underpinning regulation of the iron starvation response. Finally, we identify and characterize the frataxin homolog gene in S. pombe, pfh1. Deficiencies in frataxin provoke a neurodegenerative disease called Friedreich ataxia; the function of this protein remains controversial. We create ∆pfh1 strain as a new model system to elucidate the molecular events leading to the disease.
El peróxido de hidrógeno (H2O2) es un agente oxidante que además de participar en cascadas de señalización produce toxicidad por daño oxidativo. Parte de su toxicidad se explica por su reactividad con hierro. Así, las concentraciones de hierro en el interior celular han de estar estrictamente reguladas. Usando la levadura de fisión, Schizosaccharomyces pombe, como un sistema modelo, estudiamos las relaciones entre H2O2 y el sistema de respuesta a déficit de hierro. Genes como fep1, pcl1 y sib2, importantes para mantener su homeostasis, fueron encontrados en un análisis de 2700 mutantes de S. pombe, tras tratamiento con diferentes agentes oxidantes. Inesperadamente encontramos que H2O2 desencadena una respuesta transcripcional de déficit de hierro, incluyendo aumento de su entrada y disminución de su consumo. Ésta es una respuesta accidental debido a la sobreexpresión de proteínas como catalasa, una hemoproteína, consumidoras masivas de hierro. Encontramos además que la glutaredoxina Grx4 contiene un clúster de hierro-azufre implicado en sensar hierro. Finalmente, identificamos, caracterizamos y delecionamos el homólogo de frataxina en S. pombe, pfh1. Deficiencias en frataxina provocan ataxia de Friedreich. Los mecanismos por los cuales se desencadena esta enfermedad están todavía por elucidar, pero S. pombe es un buen sistema modelo para su estudio.

The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis.

Les champignons du genre Scedosporium sont saprophytes et pathogènes opportunistes chez l’Homme. Ces champignons présentent un tropisme particulier pour les milieux anthropisés et pollués. Plusieurs études ont révélé leur capacité à dégrader les molécules polyaromatiques issues de polluants environnementaux. Des travaux antérieurs nous ont permis de mettre en évidence que les espèces du genre Scedosporium sont capables de croître en présence de lignine. Dans l’environnement, les étapes du catabolisme des molécules polyaromatiques convergent vers un nombre limité de molécules aromatiques simples (catéchol, protocatéchuate, hydroxyquinol et gentisate) qui sont prises en charge par les voies intermédiaires centrales, également désignées sous le nom de « Fission pathways ». Une analyse bio-informatique nous a permis de caractériser les clusters de gènes dégradant ces molécules centrales chez S. apiospermum et S. aurantiacum. Des résultats expérimentaux nous ont permis de démontrer la fonctionnalité du cluster de la voie du gentisate en présence de cette molécule. Les dioxygénases qui catalysent l’ouverture du cycle benzénique, étape clé du mécanisme catabolique, sont des cibles privilégiées pour la conception de souches de délétion. Pour ce faire, la technologie CRISPR-Cas9 a été adaptée et optimisée avec succès au sein de deux souches de S. apiospermum : une souche sauvage et une souche Δku70. Pour ce faire, des protocoles différents ont été définis selon la fonctionnalité du système de réparation NHEJ. Ainsi, des souches de délétion pour le gène codant la dioxygénase ont été générées pour chacune des voies. Ces délétions impactent différemment la croissance de ces souches sur des milieux en présence des molécules centrales correspondantes. Ces résultats suggèrent, dans certaines conditions, la mise en place de mécanismes de compensation qui restent à définir. Enfin ce travail a permis d’établir pour la première fois un lien entre la dégradation des molécules aromatiques et la pathogénie d’un champignon pathogène opportuniste de l’Homme dans des expériences in vitro
Fungi of the Scedosporium genus are saprophytes, opportunistic pathogens in humans. Several studies have revealed their ability to degrade polyaromatic molecules derived from environmental pollutants. Our previous work demonstrates that species of the genus Scedosporium are able to grow in the presence of lignin. In the environment, the catabolic steps of polyaromatic molecules converge on a limited number of simple aromatic molecules (catechol, protocatechuate, hydroxyquinol and gentisate), which are handled by central intermediate pathways, also known as fission pathways. Bioinformatics analysis enabled us to characterize the gene clusters degrading these central molecules in S. apiospermum and S. aurantiacum. Experimental results demonstrate the functionality of the gentisate pathway cluster in the presence of this molecule. The dioxygenases that catalyze benzene ring opening, a key step in the catabolic mechanism, are prime targets for the design of deletion strains.To this end, CRISPR-Cas9 technology has been successfully adapted and optimized in two S. apiospermum strains: a wild-type strain and a Δku70strain. To achieve this, different protocols were defined depending on the functionality of the NHEJ repair system. Thus, deletion strains for the gene encoding dioxygenase were generated for each pathway. These deletions have a different impact on the growth of these strains on media in the presence of the corresponding core molecules. Under certain conditions, these results suggest the implementation of compensatory mechanisms that remain to be defined. Finally, this work established for the first time a link between the degradation of aromatic molecules and the pathogenesis of an opportunistic fungal pathogen of man in in vitro experiments

This thesis consists of work on the wis1 pathway: the analysis of some of the upstream components and the isolation and characterisation of genes that lie downstream of wis1. The mcs4, win1 and wis4 genes had already been shown to lie upstream of wis1. Strains were constructed with different combinations of mutations in these genes. fbp1 transcription was assayed in these strains. An additive effect was seen in win1 wis4 double mutants, suggesting that win1 and wis4 act in parallel. To identify functionally-related genes downstream of wis1, the stress sensitivity of wis1Δ cells was exploited. A screen for extragenic suppressing mutations was carried out. Several hundred heat resistant mutants were isolated. Some also presented the salt sensitivity and/or cell length defect of wisΔ. Twelve such sow (for suppressor of wis1Δ) mutants, each of which containing a single suppressing mutation, were analysed further. They fell into two linkage groups: sow1 (nine strains) and sow2 (three strains). When the sow mutations were crossed into a wis1⁺ background, both sow1 and sow2 were able to grow at temperatures above the usual range for S. pombe. In addition, sow1 strains divide at a shorter length than wild type, indicating a mitotic advance, and sow2 cells have a slightly aberrant morphology. To determine whether the sow mutations corresponded to any known genes, crosses were carried out between the sow mutants and mutants in the following genes: wis1 pathway genes (sty1, atf1, ppa1, ppa2 and ppe1), cAMP pathway genes (cyr1, pka1) cell cycle regulation genes (cdc2, cdc25, wee1, cdc13), a heat shock protein (hsp90) gene (swo1) and a gene required for maintenance of the mitotic cell cycle (pat1).

The catechol meta-fission pathway, a degradation pathway for simple aromatic compounds, is rich in enzyme chemistry and replete with structural and evolutionary diversity. Vinyl pyruvate hydratase (VPH) and MhpD catalyze the same reaction in this pathway, but in different bacterial species. These metal ion-dependent enzymes reportedly catalyze a 1,5-keto-enol tautomerization reaction followed by a Michael addition of water. MhpD, and most likely VPH, are members of the fumarylacetoacetate hydrolase (FAH) superfamily. The crystal structure of MhpD and the sequence of VPH identified four potential active site residues, Lys-60, Leu-72, Asp-78, and Ser-160 (Ser-161 in VPH). The K60A and D78N mutants of VPH and MhpD had the most damaging effects on catalysis. Moreover, the K60A mutant seemingly uncoupled tautomerization from hydration and provided evidence for an [alpha, beta]-unsaturated ketone in the reaction. The effects of the L72A and S160A (S161A in VPH) mutants were smaller, suggesting less important roles in the mechanism. 5-(carboxymethyl)-2-Oxo-3-hexene-1,6-dioate decarboxylase (COHED) is a metal ion-dependent enzyme in the homoprotocatechuate (HPC) pathway, a chromosomally encoded meta-fission pathway from Escherichia coli C that parallels the catechol meta-fission pathway. COHED is also a member of the FAH superfamily. It is a monomeric protein with two domains. It is postulated that the C-terminal domain catalyzes the decarboxylation reaction and the N-terminal domain carries out the 1,3-keto-enol tautomerization reaction. Site-directed mutagenesis, NMR, and kinetic analysis with different substrates and inhibitors have identified three potential active-site residues Glu-276, Glu-278 (in the C-terminal domain), and Lys-110 (in the N-terminal domain). Replacement of either glutamate with a glutamine eliminated both the decarboxylase and tautomerase activities. The K110A mutant also diminished both activities, but more importantly eliminated the C-3 proton/deuteron exchange reaction observed for substrate analogs. The enzymes of the catechol and homoprotocatechuate pathways provide examples of enzyme optimization toward a specific substrate even among related compounds, as reflected by the FAH superfamily. Hence, the results of these studies add to the growing body of information about how enzymes evolve and how pathways are assembled.
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Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Methanogens, members of the domain Archaea, are unique in their ability to reduce carbon substrates to methane. Coenzyme M (CoM) is required in all methanogenic pathways. The biosynthesis of this coenzyme has been well studied in Class I Methanogens, but in Class II Methanogens, such as Methanosarcina acetivorans, little is known. The first step in the biosynthetic pathway might be catalyzed by cysteate synthase (CS), which converts phosphoserine to cysteate by the addition of sulfite. The 46 kDa enzyme was successfully purified from inclusion bodies and characterized. The identity of the product was confirmed by liquid chromatography-mass spectrometry (LC-MS) results as well as by derivatization of the reaction product coupled with high pressure liquid chromatography (HPLC) analysis. Kinetic analysis showed that the enzyme has a K [subscript m] of 0.43 mM for its substrate, phosphoserine, and a K [subscript m] of 0.05 mM for its required nucleophile, sulfite. Four compounds were found to be inhibitors and IC₅₀ values were determined. The results show that CS carries out a new reaction and narrows the gap in our knowledge of Class II Methanogen CoM biosynthesis. In the second part of this dissertation, five enzymes in a newly discovered but poorly characterized pathway for the degradation of halogenated aromatic compounds in Leptothrix cholodnii SP-6 were examined. The pathway reportedly culminates in the production of 2-chloroacetaldehyde, a well-known alkylating agent. In order to determine if 2-chloroacetaldehyde is produced and how the organism survives in its presence, the pathway intermediates are being identified. To this end, 4-oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), vinylpyruvate hydratase (VPH), pyruvate aldolase (PA) and acetaldehyde dehydrogenase (AAD) were cloned, expressed and characterized. 4-OT was found to process the 5-(chloro)-2-hydroxymuconate, but only when the equilibrium was shifted by the addition of 4-OD and VPH. Steady state kinetic analysis showed that while there is a slight decrease in K [subscript m] for the halogenated substrate when compared to the non-halogenated substrate, indicating a difference in binding. There is also a 30-fold decrease in the turnover number, indicating a preference for the non-halogenated substrate. The identity of the product, 5-(chloro)-2-oxo-4-hydroxypentanoate, was verified by ¹H NMR spectroscopy. A stereochemical analysis was also carried out.
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