Translin facilitates RNA polymerase II dissociation and suppresses genome instability during RNase H2- and Dicer-deficiency

The conserved nucleic acid binding protein Translin contributes to numerous facets of mammalian biology and genetic diseases. It was first identified as a binder of cancer-associated chromosomal translocation breakpoint junctions leading to the suggestion that it was involved in genetic recombination. With a paralogous partner protein, Trax, Translin has subsequently been found to form a hetero-octomeric RNase complex that drives some of its functions, including passenger strand removal in RNA interference (RNAi). The Translin-Trax complex also degrades the precursors to tumour suppressing microRNAs in cancers deficient for the RNase III Dicer. This oncogenic activity has resulted in the Translin-Trax complex being explored as a therapeutic target. Additionally, Translin and Trax have been implicated in a wider range of biological functions ranging from sleep regulation to telomere transcript control. Here we reveal a Trax- and RNAi-independent function for Translin in dissociating RNA polymerase II from its genomic template, with loss of Translin function resulting in increased transcription-associated recombination and elevated genome instability. This provides genetic insight into the longstanding question of how Translin might influence chromosomal rearrangements in human genetic diseases and provides important functional understanding of an oncological therapeutic target

Expression of human CYP3A enzymes and drug metabolite production with fission yeast

Expression of human CYP enzymes in fission yeast and enzyme opimization by usage of polymorphisms CYP3A4*18 and CYP3A7*2. Production of 4-hydroxydiclofenac and 3-hydroxyibuprofen. CPR from Ammi majus and S. pombe as electron-transfer protein for human CYPs in the whole-cell system.Expression humaner CYP Enzyme in Spalthefe und Produktion von Wirkstoffmetabolite mit Spalthefe. Polymorphismen aus CYP3A4 und CYP3A7 wurden zur Verbesserung der Biotransformationsraten eingeführt. Produktion von 4-Hydroxydiclofenac und 3-Hydroxyibuprofen und coexpression artfremder Reduktasen im Ganzzellsystem

RNA interference-mediated co-transcriptional gene silencing in fission yeast

In the last decade or so, RNA interference (RNAi) has gained unanticipated recognition in the fields of RNA biology and gene regulation. It exists in a wide variety of eukaryotic organisms, and various forms of RNAi are involved in diverse biological processes. At its core, RNAi comprises small non-coding RNAs (sRNAs) in association with Argonaute proteins. The sRNAs are usually produced by cleavage of long double-stranded RNA by the endoribonuclease Dicer enzymes. The sRNAs guide Argonautes to target transcripts via complementary base-pairing, resulting in repression that can occur at various stages of the RNA production process. Perhaps the most well-studied mechanisms of RNAi-mediated repression are those occurring in the cytoplasm at a post-transcriptional level, whereby the target transcript is subject to degradation and/or inhibition of translation. However, well-characterised examples of nuclear RNAi also exist, and usually involve RNAi-mediated chromatin modification such as DNA methylation in plants and histone methylation in protozoa and fungi. These modifications can contribute to heterochromatin formation and inhibit RNA production at the level of transcription. In addition to mediating post-transcriptional and transcriptional gene silencing, recent evidence from several organisms suggests that RNAi can mediate co-transcriptional gene silencing (CTGS), whereby physical association of the RNAi machinery with chromatin can promote degradation of the nascent transcripts and/or inhibit transcription. Such a mode of silencing was first proposed in the fission yeast Schizosaccharomyces pombe (S. pombe), where the RNAi machinery is thought to repress heterochromatic RNA at a transcriptional and co-transcriptional level. During my PhD, I focused on the association of the RNAi machinery with chromatin in S. pombe. Using a sensitive chromatin profiling technique called DamID, I was able to provide the first direct evidence that S. pombe Dicer functions in cis on chromatin. Secondly, I uncovered a novel role for RNAi in gene regulation outside of the well-studied heterochromatic regions. The evidence presented here shows that the S. pombe RNAi machinery is concentrated at nuclear pores where it acts to co-transcriptionally degrade euchromatic RNAs, particularly those from retrotransposon long-terminal repeats, non-coding RNAs and stress response genes bound by the activating transcription factor Atf1. This may keep such features ‘poised’ for expression, allowing more rapid upregulation under inducing conditions. Of particular note, Argonaute is not required for targeting the other RNAi components to euchromatin, suggesting that in this case guidance by the sRNA is not responsible for recognition of substrates. I discuss the implications of these results, particularly in the context of RNAi in other eukaryotes

Caractérisation moléculaire et fonctionnelle de Cif1p, une protéine orpheline impliquée dans le phénomène épigénétique de viabilité de la levure S. pombe en absence de la chaperone calnexine

Le repliement des protéines est un processus cellulaire crucial impliquant plusieurs protéines dont la calnexine, une chaperone du réticulum endoplasmique. Notre laboratoire et un autre groupe avons démontré que la calnexine est essentielle à la viabilité de la levure Schizosaccharomyces pombe. Dans le cadre d’études structure-fonction portant sur cette protéine, nous avons découvert un phénomène permettant la viabilité des cellules en absence de la calnexine. Cet état, nommé Cin pour calnexine independence, est induit par un mutant de la calnexine dépourvu du domaine central hautement conservé (Δhcd_Cnx1p). La caractérisation de l’état Cin a révélé plusieurs caractéristiques particulières telle la dominance, sa transmission de façon non-Mendélienne à la progéniture méïotique et sa transmission par des extraits protéiques dépourvus d’acides nucléiques. Toutes ces propriétés suggèrent donc que l’état Cin est médié via un élément de type prion. Le gène cif1+, pour calnexin independence factor, a été isolé lors de criblages visant à identifier des gènes impliqués dans l’état Cin. Il encode pour une protéine orpheline dont la surexpression induit de façon stable un état de viabilité en l’absence de la calnexine. Cet état diffère génétiquement et phénotypiquement de l’état Cin induit par le mutant Δhcd_Cnx1p préalablement caractérisé, ce qui suggère deux voies parallèles de signalisation du phénomène Cin. Une caractérisation exhaustive de Cif1p a permis de démontrer qu’il ne s’agissait pas du prion responsable de l’état Cin, malgré que cette protéine possède certaines propriétés typiques des prions in vitro. Finalement, Cif1p est une protéine nucléolaire dont la bonne localisation est essentielle à sa capacité à induire l’état Cin. Ceci suggère une interaction entre la fonction essentielle de la calnexine et une fonction exécutée dans le nucléole. Lors d’études visant à élucider la fonction cellulaire de Cif1p, il a été établi qu’elle interagissait avec certaines protéines de la grosse sous-unité du ribosome telle la protéine L3. Cependant, Cif1p ne co-sédimente pas avec des sous-unités ribosomales assemblées, des ribosomes ou des polysomes. De plus, des cellules contenant une délétion génomique de cif1 voient leur contenu en ribosomes perturbé lors de la phase stationnaire. Il semble donc que Cif1p joue un rôle dans la biosynthèse des ribosomes lors de la phase stationnaire. Ce rôle spécifique à cette phase de croissance coincide avec un clivage de la portion N-terminale de Cif1p, clivage qui a lieu lors de l’entrée des cellules en phase stationnaire. De plus, des études effectuées récemment dans notre laboratoire proposent que la calnexine joue un rôle important dans la signalisation de l’apoptose, et ce particulièrement en phase stationnaire. Ainsi, une voie impliquant Cif1p, sa fonction nucléolaire dans la biosynthèse des ribosomes en phase stationnaire, la calnexine et la médiation de l’apoptose semble se dessiner. D’autres travaux, notamment sur la fonction exacte de Cif1p, le rôle de son clivage et les autres composantes impliquées dans le phénomène Cin nous permettront de dessiner un portrait plus complet de cette voie cellulaire inédite.Protein folding is a vital process that involves many proteins of the cell. One of them is calnexin, a chaperone of the endoplasmic reticulum. In the fission yeast Schizosaccharomyces pombe, calnexin is essential for survival of the cells. During structure-function studies on calnexin, our laboratory discovered a phenomenon allowing the viability of cells without this chaperone. This state, designated Cin for Calnexin INdependence, is induced by a calnexin mutant devoid of the highly conserved central domain (Δhcd_Cnx1p). Characterization of the Cin cells showed several exceptional properties such as dominance, non-Mendelian transmission and transmission via cell extracts devoid of nucleic acids of the Cin state. All these observations suggested that the Cin phenomenon is mediated via a prionic element. To identify genes implicated in the Cin state, genetic screens were performed. They led to the identification of the cif1+ gene, for calnexin independence factor. This gene encodes an orphan protein, the overexpression of which stably induces a state of viability in the absence of calnexin. Notably, this state is genetically and phenotypically distinct from the previously isolated Cin state arising from Δhcd_Cnx1p expression. This suggests the presence of two parallel pathways both able to signal the induction of the Cin phenomenon. The exhaustive characterization of Cif1p showed that it is not the prion solely responsible for the Cin state, although it displays prion-like properties in vitro. Finally, nucleolar localization of Cif1p is required to induce the Cincif1 state, thus suggesting an unexpected interaction between the vital cellular role of calnexin and a function of the nucleolus. While investigating Cif1p function in the cell, we observed that it interacts with ribosomal proteins of the large subunit, notably L3, but it does not sediment with assembled ribosomal subunits or whole ribosomes. However, cells containing a genomic deletion of cif1 also have a disrupted ribosome content during stationary phase. Altogether, these results suggest that Cif1p has a role in ribosomal biogenesis during stationary phase. This growth-phase specific role correlates with the occurence during stationary phase of a cleavage in the N-terminal part of Cif1p. Recent studies from our laboratory proposed that calnexin plays an important role in apoptosis signaling, especially in stationary phase. Thus, a pathway implicating Cif1p, its nucleolar function in ribosome biosynthesis in stationary phase, calnexin and apoptosis signaling is starting to emerge. However more studies, notably on the exact function of Cif1p, the role of its cleavage and the other proteins implicated in the Cin state will be necessary to draw the complete scheme of this unprecedented cellular pathway

Functional Roles of Nucleases in DNA Metabolism and Genome Stability

The work outlined in this dissertation focuses on two distinct areas that are important for genome stability. Both areas focus on DNA repair pathways that require the action of nucleases, specifically Exonuclease 5 and Ribonuclease H2. First, I describe the biochemical and molecular characterization of the novel Exonuclease 5 family of enzymes from S. cerevisiae, S. pombe, and humans. The Exo5 family consists of bi-directional single-strand DNA specific exonucleases that all contain an iron-sulfur cluster as a structural motif and all have various roles in DNA metabolism. In the Saccharomycetales order that includes the budding yeast, S. cerevisiae, Exo5 is a mitochondrial protein that is essential for mitochondrial genome maintenance. In an unrelated yeast species, Schizosaccharomyes pombe, Exo5 is important for both nuclear and mitochondrial DNA metabolism. The human ortholog is important for nuclear genome stability, and for DNA repair. The work outlined in Chapter II of this Dissertation establishes Exo5 as a protein that is important for DNA metabolism. The second area of study outlined in Chapters III and IV is related to the phenomenon of ribonucleotide incorporation into the genome by replicative polymerases, and these chapters focus on the enzymes that remove these noncanonical nucleotides. Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. In Chapter III, I present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. I highlight the requirement for RNase H2 in the process of RER and investigate the redundancies at different steps of repair. Also outlined in this dissertation is the dissection of the different functions of RNase H2 in RER and in the removal of RNA-loops in DNA, and implications for genome instability in human diseases that are affected for these activities. Chapter IV of this dissertation discusses work on an alternative pathway for ribonucleotide removal from the genome by Topoisomerase I. In S. cerevisiae, deletion of rnh201, the catalytic subunit of RNase H2, results in the persistence of ribonucleotides remain in the genome, which leads to ~100-fold increase in the frequency of 2-5 bp deletions at di-nucleotide repeat sequences. These deletions are dependent on topoisomerase I (Top1) activity. Here we present an in vitro reconstitution of the mechanism of Top1-dependent deletions at di-nucleotide repeat sequences and a mechanism for Top1-initiated removal of ribonucleotides outside of the context of these repeat sequences in S. cerevisiae. Top1 attack at a ribonucleotide leads to the formation of a 2\u27, 3\u27 cyclic phosphate terminated ssDNA nick, followed by subsequent formation of a Top1-cleavage complex (Top1-cc) upstream of the 2\u27, 3\u27 cyclic phosphate. If the ribonucleotide is in the context of a di-nucleotide repeat, there can be realignment of the DNA allowing for religation and release of Top1, leading to a 2-nucleotide deletion. If the ribonucleotide resides outside a repeat sequence, the realignment is not possible and a different pathway must repair the Top1-cc. Tdp1-dependent repair of Top1-cc requires prior proteolytic processing of the Top1-cc before it can be removed leaving a 3\u27-phosphate that can be removed by Tpp1, Apn1, or Apn2 forming a substrate suitable for repair by DNA polymerase δ, FEN1 and DNA ligase

Protein-protein interactions of the DNA polymerase ð complex in the fission yeast Schizosaccharomyces pombe

In eukaryotes there are three essential DNA polymerases that are involved in the bulk o f DNA replication: pola, polS and pole. P ola is involved in generating a short RNA-DNA primer. PolS and 8 are involved in the elongation process o f DNA replication. It has been suggested that polS is the key enzyme that performs all o f the processive DNA replication since the catalytic domains o f pole are not essential.In S. pombe polδ is comprised of four subunits: Pol3- the catalytic or A subunit, Cdcl- the B subunit, Cdc27- the C subunit, and Cdml- the D subunit. Polδ in S. cerevisiae and mammals have homologues o f these subunits, except for the D subunit of which there is no homologue in S. cerevisiae.In this thesis polS from S. pombe has been studied in two ways. One approach was to investigate the protein-protein interactions within polδ, and the other was to investigate Cdcl, the highly conserved B subunit of unknown function. The proteinprotein interactions were investigated using a combination of two-hybrid assays and mutational analysis. Cdcl was investigated by performing extensive mutational analysis using both random and site directed methods.The combination of approaches has demonstrated that the C- terminal ZnF2 region of both S. pom be and S. cerevisiae A subunit (Pol3) is involved in the direct binding to the B subunit. The four cysteines present in the zinc finger are involved in maintaining the structure of both S. pombe and S. cerevisiae ZnF2. Mutational analysis o f Cdcl (the S. pombe B subunit) has identified a conserved region (DomIII) that could be involved in the function of Cdcl. Additionally, binding assays with the Cdcl mutants have suggested a region of Cdcl (from amino acids 293 to 329) as being involved in the binding to Pol3