Rozprawy doktorskie na temat „Ribosomal RNA (rRNA)”

The molecular characterization of twenty six Theileria spp. isolates and one C. felis isolate were done on the small subunit ribosomal RNA (SSU rRNA) gene, the 5.8S gene, and the two internal transcribed spacer regions using gDNA. The SSU rRNA gene is increasingly accepted as a widely used marker for characterization, taxonomic classification, and phylogenetic analysis and this gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (Chae et al. 1998; Chae et al., 1999 a,b,c; Stockham et al., 2000; Cossio-Bayugar et al., 2002; Gubbels et al., 2000). The genomic region consists of the internal transcribed spacer 1 (ITS 1), the 5.8S gene, and internal transcribed spacer 2 (ITS 2) (ITS 1-5.8S-ITS 2 gene region) and separates the SSU rRNA gene from the large subunit ribosomal RNA gene. The 5.8S rRNA gene is highly conserved in size and nucleotide sequence, is relatively constant in molecular weight, and has an average chain length of approximately 160 nucleotides and has proven useful in dividing subgenera of Gyrodactylus ((Nazar, 1984; Zietara et al., 2002). Pairwise comparisons were done between the clones of an individual isolate and among the clones of the different isolates. Phylogenetic trees were made from the resulting sequences. This study shows that different SSU rRNA genes may be associated with ITS 1-5.8S-ITS 2 gene regions of distinct sequence in the same isolate. This study also demonstrates that considerable ITS 1-5.8S-ITS 2 gene region sequence variation may exist within a species. This may be useful for subspeciation designation, or may simply reflect considerable variation within the population. This study shows that the ITS 1-5.8S-ITS 2 gene region may be a useful molecular marker for the taxonomy of Theileria spp.

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

The first high resolution crystal structures initiated a new era in ribosomal studies in which investigations depend on knowledge of atomic coordinates. The studies I conducted on the three-dimensional structure of the ribosome aim to reveal and understand the ribosome modular organization, and at the same time to investigate protein-RNA interaction mechasisms. All the results presented in this thesis refer to the case study of the large ribosomal subunit 50S of Haloarcula marismortui, omitting the 5S rRNA. Three-dimensional RNA motifs are the basic building blocks of ribosomal RNA (rRNA) architecture. A novel method to search for these substructures, based on shape histograms, is presented in Chapter 2. The shape histogram is a vector representation of the distribution of distances between a set of points and a fixed one. Shape histograms are not only an efficient tool to compare RNA fragments, but also they well capture the structural similarities behind the flexible and highly variable structure of ribosomal RNA. The method outperforms existing ones in time efficiency, reaching the same level of correctness. Branching points of three or more helices, named junctions, are the most variable and complex loops of rRNA, and the least characterized. Shape histograms together with angles between inter-helical fragments provide major informations on the 3D conformation of junctions: angles give informations on the eccentricity of the loop, while shape histogram range and distribution reveal the folding. Based on these features I propose a possible classification of these motifs with respect to their 3D conformation. Finally in Chapter 4 I dissect protein-RNA interactions within the ribosome. Statistical analysis reveal distinguishing features of the ribosome with respect to protein interactions with other RNA molecules, such as the dominant role of the ribose group. Furthermore protein interaction mechanisms with known RNA motifs are investigated, in particular with standard tetraloops, kink-turns, and single extruded nucleotides in general. A consensus interaction pattern is detected for protein contact surfaces with standard tetraloops, characterized by dense areas of contacts made by positive residues (mainly arginine). Ribosomal proteins also reveal a characteristic binding site with kink-turns in correspondence of the extruded base. Due to its shape, this site has been called tripod. Tripods proved to be common to several single extruded nucleotides. Besides a conserved shape, tripods show a preference towards purines, especially adenine, and typically make hydrogen bonds.
Le prime strutture cristallizate ad alta risoluzione diedero inizio ad una nuova era negli studi sui ribosomi, era nella quale la ricerca beneficia delle coordinate spaziali di ogni singolo atomo. Gli studi che ho condotto sulla struttura tridimensionale del ribosoma puntano a compredere l’organizzazione modulare della molecola, ed allo stesso tempo ad investigare i meccanismi di interazione proteine-RNA. Tutti i risultati presentati in questa tesi riferiscono alla subunità principale 50S di Haloarcula marismortui, omettendo il filamento di RNA 5S. Motivi tridimensionali ricorrenti sono i mattoni basilari dell’architettura dello RNA ribosomiale: essi sono caratterizzati da una struttura 3D conservata, e ricorrono frequentemente all’interno della molecola. Un nuovo metodo per ricercare queste sotto strutture, basato su shape histogram, è presentato al Capitolo 2. Lo shape histogram è una rappresentazione vettoriale della distribuzione delle distanze di un insieme di punti da uno fissato. Gli shape histogram non sono solo strumenti efficienti per confrontare frammenti diversi di RNA, sono inoltre in grado di catturare le similarità strutturali nascoste dalla struttura flessibile ed altamente variabile dello RNA ribosomiale. Il metodo infatti si dimostra più efficiente di quelli noti in letteratura, ed ugualmente efficace in termini di correttezza dei risultati. Tra i loop studiati, le giunzioni di tre o più eliche sono il tipo più variabile e complesso, ed il meno caratterizzato. Gli shape histogram insieme alle sequenze di angoli formati dai frammenti congiungenti più eliche sono in grado di dare molte informazioni sulla conformazione tridimensionale di queste giunzioni: gli angoli forniscono indicazioni sull’eccentricità di una giunzione, mentre range e distribuzione dei valori degli shape histogram rivelano il folding. Basandosi su queste caratteristiche, propongo una classificazione per questi motivi rispetto alla loro conformazione nello spazio. Infine il Capitolo 4 esamina le interazioni proteine-RNA all’interno del ribosoma. Osservazioni di tipo statistico rivelano caratteristiche distintive del ribosoma rispetto alle interazioni proteiche con altri tipi di RNA, come ad esempio il ruolo dominante del gruppo ribosio. Inoltre vengono studiati i meccanismi di interazione delle proteine con i motivi strutturali, in particolare standard tetraloop, kink-turn e basi esposte. Un pattern comune di interazioni è rintracciato per le superfici di contatto formate dalle proteine con i tetraloop, caratterizzato da zone dense di interazioni fatte da aminoacidi a carica positiva (principalmente arginine). Le proteine ribosomiali rivelano inoltre un sito di contatto caratteristico nelle interazioni con i kink-turn, in corrispondenza della base esposta. Per la sua forma questo sito è stato chiamato tripod, ovvero tripode. I tripodi si sono dimostrati essere in realtà comuni a molti nucleotidi non accoppiati la cui base è esposta. Oltre ad una conformazione tridimensionale conservata, i tripodi dimostrano una preferenza verso le purine, soprattutto adenina, e tipicamente formano legami idrogeno.

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the 30S ribosomal subunit, has been traced to the arm/rmt family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosylmethionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA in the 30S ribosomal subunit. Neither 16S rRNA alone nor intact 70S ribosome is an efficient substrate for armA methyltransferase. To more fully characterize this family of enzymes, xiii we have investigated the substrate requirements of ArmA. We determined the Mg2+ dependence of ArmA activity and found that the enzyme could recognize both translationally active and translationally inactive forms of 30S subunits. To identify the site of interaction between ArmA and the 30S subunit, we used hydroxyl radical cleavage of 16S rRNA mediated by ferrous iron chelated to several sites on the ArmA molecule that were mutated to cysteine. This data suggests that significant conformational changes in 30S structure are involved in binding of ArmA. We hypothesized that a precursor intermediate in the biogenesis of the 30S subunit might be the optimal substrate for ArmA enzymes in vivo. To test this, we prepared 30S particles partially depleted of proteins by treatment with increasing concentrations of LiCl and assayed them for ArmA methylation. Even low concentrations of LiCl alter the 30S particles and greatly diminish their susceptibility to methylation. Additionally, a previously identified pre-30S particle isolated from an E. coli culture was assayed for its ability to support methylation by ArmA and found to be inferior to intact 30S particles as a methylation substrate. Thus, testing of immature particles prepared from in vitro and in vivo sources suggest that ArmA works very late in the 30S biogenesis pathway. Initiation factor 3 (IF3), a factor that only binds fully mature 30S particles, does not inhibit the ArmA methylation, while kasugamycin methyltransferase (KsgA) abolishes ArmA activity by sharing the same binding site with ArmA. From aforementioned experiments, we conclude that ArmA is most active toward 30S ribosomal subunits that are at or very near full maturation.

La synthèse des protéines cellulaires requiert à la fois des ribosomes fonctionnels et des ARN de transfert (ARNt) matures comme molécules adaptatrices. Les ribosomes sont de larges complexes ribonucléoprotéiques dont la biogenèse représente la plupart de la transcription cellulaire et consomme une majeure partie de l’énergie de la cellule. Par conséquent, la biogenèse des ribosomes fait l’objet d’une régulation importante afin d’ajuster le nombre de ribosomes aux besoins de la cellule et de dégrader efficacement les particules défectueuses qui pourraient interférer avec la traduction. Les ARNs ribosomiques (ARNr) et les ARNt sont tous deux transcrits sous formes de précurseurs et sont universellement maturés pour devenir fonctionnels pour la traduction. Ce travail de thèse a permis de mettre en évidence un couplage entre la maturation des ARNt et la biogenèse des ribosomes chez la bactérie modèle à Gram positif Bacillus subtilis. Ainsi, l’accumulation d’ARNt immatures lors d’une déplétion en enzymes de maturation, abolit spécifiquement la maturation en 3’ de l’ARNr 16S par l’endoribonucléase YqfG/YbeY, dernière étape dans la formation de la petite sous-unité ribosomique (30S). Nous avons mis en évidence que ce défaut de maturation résultait d’un défaut d’assemblage tardif du 30S coïncidant avec des changements d’expression de plusieurs facteurs d’assemblage du ribosome. Nous avons montré que cette modulation d’expression provenait d’effets transcriptionel et post-transcriptionel. De façon inédite, nos résultats indiquent que l’accumulation d’ARNt immatures est perçue par RelA (le facteur de la réponse stringente), déclenchant la production de (p)ppGpp. Nous avons observé que cette synthèse de (p)ppGpp et la baisse concomitante des niveaux de GTP cellulaire, inhibe la maturation de l’ARNr 16S en 3’, probablement via un blocage des GTPases impliquées dans l’assemblage des ribosomes. L’inhibition de la maturation de l’ARNr 16S côté 3’ est supposée conduire, par la suite, à une dégradation des particules partiellement assemblées par la RNase R. Ainsi, nos résultats supportent un modèle où RelA jouerait un rôle central ; en percevant une déficience de maturation des ARNt et en ajustant, en conséquence, la biogenèse des ribosomes via la production de (p)ppGpp. Ce mécanisme de couplage permettrait de maintenir un équilibre fonctionnel entre ARNt et ARNr, les deux composants majeurs de la machinerie de traduction
Cellular protein synthesis both requires functional ribosomes and mature transfer RNAs (tRNAs) as adapter molecules. The ribosomes are large essential ribonucleoprotein complexes whose biogenesis accounts for most of cellular transcription and consumes a major portion of the cell’s energy. Ribosome biogenesis is therefore tightly adjusted to the cellular needs and actively surveilled to rapidly degrade defective particles that could interfere with translation. Interestingly, tRNAs and ribosomal RNAs (rRNAs) are both transcribed from longer primary transcripts and universally require processing to become functional for translation. In this thesis, I have characterized a coupling mechanism between tRNA processing and ribosome biogenesis in the Gram-positive model organism Bacillus subtilis. Accumulation of immature tRNAs during tRNA maturase depletion, specifically abolishes 16S rRNA 3’ processing by the endonuclease YqfG/YbeY, the last step in small ribosomal subunit formation. We showed that this maturation deficiency resulted from a late small subunit (30S) assembly defect coinciding with changes in expression of several key 30S assembly cofactors, mediated by both transcriptional and post-transcriptional effects. Interestingly, our results indicate that accumulation of immature tRNAs is sensed by the stringent factor RelA and triggers (p)ppGpp production. We showed that (p)ppGpp synthesis and the accompanying decrease in GTP levels inhibits 16S rRNA 3’ processing, most likely by affecting GTPases involved in ribosome assembly. The inhibition of 16S rRNA 3’ processing is thought to further lead to degradation of partially assembled particles by RNase R. Thus, we propose a model where RelA senses temporary slow-downs in tRNA maturation and this leads to an appropriate readjustment of ribosome biogenesis. This coupling mechanism would maintain the physiological balance between tRNAs and rRNAs, the two major components of the translation machinery

Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli. RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V. RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity. These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Δ cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Δ cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.

Culicoides (Diptera: Ceratopogonidae) are small (<3mm) blood feeding flies. These flies are biological vectors of viruses, protozoa and filarial nematodes affecting birds, humans, and other animals. Among the viruses transmitted those causing bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease (EHD) are of major veterinary significance. Culicoides (Avaritia) imicola Kieffer, a proven vector of both AHS and BT viruses, is the most abundant and wide spread livestock-associated Culicoides species in South Africa. Field isolations of virus and oral susceptibility studies, however, indicated that a second Avaritia species, C. bolitinos Meiswinkel may be a potential vector of both BT virus (BTV) and AHS virus (AHSV). Differences in oral susceptibility, which are under genetic control, of populations from different geographical areas to viruses may be an indication of genetic differences between these populations, which may be the result of limited contact between these populations. A good knowledge of the distribution, spread and genetic structure of the insect vector is essential in understanding AHS or BT disease epidemiology. In the present study, an effort was made to gather field specimens of both C. imicola and C. bolitinos from different areas within their natural distribution in South Africa. The aim was to partially sequence two mitochondrial genes from these specimens and to analyse the sequence data making use of phylogenetic trees to clarify the genetic relationships between individuals or groups collected from geographically distinct sites. The two species were collected from four geographically separated areas in South Africa viz. Gauteng Province, Eastern Cape Province, Western Cape Province as well as the Free State Province. DNA was extracted from a total of 120 individual midges of the two Culicoides species using DNA extraction kits. Extracted DNA was analysed using PCR, sequencing as well as phylogenetic methods. A total of 117 mitochondrial DNA COI and 104 mitochondrial 16S ribosomal RNA CulidoidesDissertation (MSc)--University of Pretoria, 2010.
Veterinary Tropical Diseases
unrestricted

La biogenèse des ribosomes, aussi appelée la synthèse ribosomale, est un processus cellulaire important se déroulant dans le nucléole et implique la transcription par les trois ARN polymérases nucléaires. L’étape initiale et limitante de ce processus est la transcription des ARNs ribosomaux catalytiques, 28S, 18S and 5.8S, sous la forme d’un long précurseur d’ARN ribosomal (pre-ARNr/47S) par l’ARN polymérase I (RPI). RPI possède un ensemble de facteurs de transcription généraux responsables de son activation. Ces facteurs sont la protéine architecturale UBF, le facteur SL1 qui contient TBP, le facteur d’initiation RRN3 et le facteur de terminaison TTF1. La synthèse de l’ARN ribosomale est finement régulée et correspond à 30-50% de l’ensemble de la transcription de la cellule. De plus, ce processus est lié à la croissance cellulaire, la transformation, la prolifération et à l’activité des facteurs suppresseurs de tumeurs et des oncogènes. UBF et RRN3 sont notamment activés par plusieurs voies de signalisation de croissance cellulaire. Dans les cellules de mammifère, il existe ~200 copies d’ADNr par génome haploïde. Les fragments répétés d’ADN ribosomal sont arrangés en répétition en tandem sur les bras courts des chromosomes acrocentriques. De façon intéressante, dans les cellules somatiques, seulement la moitié des copies d’ADNr sont actives, alors que les autres sont maintenues dans une forme inactive par les modifications épigénétiques et la formation d’hétérochromatine. La raison pour laquelle le génome contient autant de copies et la régulation de leur activité ne sont pas bien comprises. Cette thèse présente l’analyse de l’importance in vivo d’UBF et de RRN3 pour la régulation de la transcription de l’ARNr et pour le maintien de la structure chromatinienne de l’ADNr. Nous avons précédemment analysé la perte de fonction de UBF dans les fibroblastes embryonnaires de souris en utilisant le système de perte de fonction conditionnelle dépendante du tamoxifène. Puisque l’un de nos objectifs était de comparer la fonction de RRN3 dans un modèle similaire, nous avons réanalysé la perte de fonction de RRN3 chez la souris et généré des lignées cellulaires comme préalablement réalisées avec la perte de fonction d’UBF. Nous avons déterminé que RRN3 est essentiel à la préimplantation et le développement est arrêté à E3.5, ce qui contredit les résultats obtenus par un autre groupe qui avait obtenu un arrêt du développement beaucoup plus tardif, à E9.5. Une lignée de fibroblastes embryonnaires de souris inductible au tamoxifène a été créée pour RRN3 de façon similaire à ce qui avait été fait pour UBF. La perte de fonction d’UBF ou de RRN3 inhibe la transcription par RPI. Par contre, nous démontrons que UBF est responsable du recrutement à l’ADNr des autres facteurs associés à RPI et du maintient de l’état ouvert de la chromatine. En comparaison, RRN3 est requis simplement pour le recrutement de RPI. Dans cette étude, nous avons également identifié une région frontalière en amont de l’ADNr formée de H2A.Z, TTF1, CTCF et des modifications d’histones activatrices. Nous avons également découvert que la perte d’UBF entraine une mort cellulaire synchronisée par apoptose, indépendamment de p53 et ce spécifiquement dans les lignées cellulaires transformées. Ce résultat suggère qu’il pourrait être possible de cibler UBF dans le traitement contre le cancer puisque la perte de UBF dans les lignées cellulaires primaires cause un arrêt de prolifération sans entrainer l’apoptose. Finalement, nous avons observé que le niveau d’activité de l’ADNr dans les cellules pluripotentes est différent que dans les cellules différenciées. Des lignées de cellules souches embryonnaires (ESCs) ont été générées à partir des souris conditionnelles pour UBF et RRN3 et nos résultats préliminaires suggèrent que la totalité des gènes de l’ADNr est active dans les cellules pluripotentes. Ce modèle est idéal pour étudier la régulation de l’ADNr ainsi que le rôle de UBF et RRN3 dans cette régulation après l’induction de la différentiation. En résumé, ces résultats permettront de clarifier le rôle in vivo de UBF et RRN3 dans la transcription de l’ARN ribosomal et dans le maintien de l’intégrité de l’ADNr.
Ribosome biogenesis, or the synthesis of ribosomes, is an important cell process occurring in the nucleolus that utilizes transcription by all three nuclear RNA polymerases. The initial and rate-limiting step is the transcription of the catalytic ribosomal RNAs 28S, 18S and 5.8S in the form of a precursor ribosomal RNA (pre-rRNA/47S) by RNA polymerase I (RPI, also known as Pol1 and POLR1). RPI has a dedicated set of basal factors responsible for its activation. These are the architectural factor UBF, the TBP containing factor SL1, the initiation factor RRN3, and the termination factor TTF1. Ribosomal RNA synthesis is tightly regulated and accounts for 30-50% of total gene transcription. As such, this process is linked to cell growth, transformation, proliferation and the actions of tumour suppressors and oncogenes. Notably, UBF and RRN3 are activated by many of the same growth signaling pathways. The human and mouse haploid genome contain ~200 copies of the ribosomal RNA genes, the ribosomal DNA (rDNA). These ribosomal DNA copies are arranged in tandem repeats on the short arms of acrocentric chromosomes. Interestingly, only a fraction of the rDNA copies are active, and a significant number are epigenetically silenced and heterochromatic. The reason for having so many copies and their regulation in vivo by silencing is not yet understood, though it has been connected with genome stability. This thesis presents the analysis of the in vivo requirements for UBF and RRN3 in rRNA transcription and rDNA chromatin structure. We had previously analyzed the loss of UBF in mouse embryonic fibroblasts using tamoxifen-dependent conditional knockout. As we wanted to compare the loss of RRN3 in a similar model, we re-analyzed the RRN3 knockout mice and created cell lines as was performed for the UBF knockout. Importantly, we find that RRN3 is essential for preimplantation and its loss arrests development at E3.5, contrary to previous work that showed a late E9.5 developmental arrest. Using mouse embryonic fibroblast (MEF) cell lines conditional for UBF or RRN3, we found that the loss of either factor prevented RPI transcription. However, we found that UBF was essential for the recruitment of the other RPI transcription factors and the formation of the preinitiation complex, as well as to maintain an open rDNA chromatin structure, while RRN3 was required only for RPI recruitment. These studies allowed us to identify an upstream boundary element on the rDNA formed of H2A.Z, TTF1, CTCF and activating histone marks, which is independent of RPI activity. We also found that UBF loss, but not RRN3 loss, led to a synchronous and massive p53-independent apoptosis, specifically in oncogenically transformed cells. This strongly suggests that drug targeting UBF could be a viable cancer treatment. Finally, we have observed that the rDNA activity status in pluripotent cells differs from that of differentiated cells. Embryonic stem cells (ESCs) were also generated from the mice conditional for UBF and RRN3. Preliminary results indicate that, unlike somatic cells, all the rRNA genes in these and other pluripotent cell lines are potentially active. This makes ESCs and their differentiation an ideal model in which to study the establishment of rDNA silencing and the role of UBF and/or RRN3 in this process. Together these data define the in vivo roles of UBF and RRN3 in ribosomal RNA transcription and suggest mechanisms by which they maintain rDNA integrity and may drive cell differentiation.

Em eucariotos, o processamento de pré-rRNA depende de vários fatores como endonucleases, exonucleases, RNA helicases, enzimas modificadoras de rRNA e componentes de snoRNPs. Com o objetivo de caracterizar novas proteínas envolvidas no processamento de pré-rRNA, foi identificada a proteína Nop53p interagindo com a proteína nucleolar Nop17p a partir de uma varredura da biblioteca de cDNAs de Saccharomyces cerevisiae. A cepa condicional contendo a seqüência da ORF NOP53 sob controle do promotor de galactose não cresce em meio contendo glicose, indicando que Nop53p seja uma proteína essencial para a viabilidade celular. Os resultados deste trabalho demonstram que Nop53p está envolvida nas etapas iniciais de clivagem do pré-rRNA, assim como nas clivagens responsáveis pela formação dos rRNAs maduros 5.8S e 25S. Análise mais detalhada do processamento de pré-RNA por Northern blot e \"pulse-chase labeling\", revelou também que Nop53p afeta principalmente o processamento do rRNA intermediário 27S, que origina os rRNAs maduros 5.8S e 25S. Nop53p participa do processamento desses rRNAs afetando a poliadenilação dos precursores dos rRNAs 5.8S e 25S. Experimentos de co-imunoprecipitação de RNA com a proteína de fusão ProtA-Nop53p confirmaram o envolvimento de Nop53p no processamento do 27S rRNA, indicando que essa proteína possa ligar RNA diretamente. A capacidade de Nop53p de ligar RNA foi confirmada através de testes in vitro, enquanto que ensaios de co-imunoprecipitação de cromatina revelaram que Nop53p liga-se ao rRNA 5.8S durante a transcrição. Nop53p regula a função do exossomo através da sua interação direta com a subunidade exclusivamente nuclear deste complexo, Rrp6p.
In eukaryotes, the rRNA processing depends on several factors, such as, endonucleases, exonucleases, RNA helicases, rRNA modifying enzymes and components of the snoRNPs. With the purpose of characterizing new proteins involved in pre-rRNA processing, Nop53p was identified interacting with the nucleolar protein Nop17p in a two hybrid assay. The conditional yeast strain containing the sequence of the ORF NOP53 under the control of the galactose promoter cannot grow in medium containing glucose, indicating that the protein is essential for cell viability. The results of this work demonstrate that Nop53p is involved in the initial steps of pre-rRNA processing and in the cleavages responsible for the formation of the mature rRNAs 5.8S and 25S. A more detailed analysis of the pre-rRNA processing, by Northern blot and pulse-chase labeling, revealed that Nop53p affects the processing of the 27S precursor, that originates the rRNAs 5.8S and 25S. Nop53p participates in the processing of these RNAs by affecting the polyadenylation of the precursors of the rRNAs 5.8S and 25S. RNA co-imunoprecipitation assays with the fusion protein A-Nop53p confirmed the involvement of Nop53p in the processing of the 27S pre-rRNA, indicating that the protein may interact directly with the RNA. The capacity of Nop53p to bind RNA was confirmed by in vitro assays, while chromatin imunoprecipitation assays demonstrated that Nop53p binds the 5.8S rRNA co- transcriptionally. Nop53p regulates the function of the exosome by interacting directly with the exclusively nuclear subunit of the complex, Rrp6p.

La synthèse du ribosome est un processus compliqué, très hiérarchisé et essentiel à toutes les cellules vivantes. La complexité de ce processus tient notamment au fait que les différentes étapes de la biogenèse du ribosome eucaryote sont temporellement et spatialement organisées dans des compartiments cellulaires différents (le nucléole, le nucléoplasme et le cytoplasme). Il est toutefois connu que le pré-ARNr 35S (le précurseur de trois des quatre ARNr, les ARNr 18S, 5.8S et 25S) est pris en charge dès sa synthèse par des facteurs impliqués dans sa maturation. Ainsi, la formation d’un ribosome requiert l’association, sur le transcrit naissant, des facteurs de synthèse, au nombre de 400. Ces facteurs essentiels interagissent transitoirement avec l’ARNr et ne font pas partie des particules ribosomiques matures impliquées dans la traduction. Leur rôle est d’assister le remodelage constant du pré-ribosome et le processus d’assemblage des sous-unités.

Parmi ces facteurs de synthèse, nous avons caractérisé en détail, chez la levure et chez l’homme, la protéine Las1 impliquée dans la maturation des deux extrémités de l’ITS2, séquence qui sépare les ARNr 5.8S et 25S/28S. Chez la levure, en absence de la protéine Las1, les analyses de profils de polysomes révèlent un déficit de sous-unité 60S et l’apparition d’« halfmères ». Les techniques de purification d’affinité et de gradient de sédimentation nous indiquent que Las1 est associée aux pré-ribosomes 60S et qu’elle interagit avec de nombreux facteurs de synthèse de la petite, de la grande sous-unité ou des deux. De plus, Las1 copurifie avec des pré-ribosomes qui contiennent aussi les exoribonucléases 5’-3’ Rat1/Rai1 et Xrn1. Rai1 coordonne la maturation aux deux extrémités de l’ARNr 5.8S. Nous suggérons que Las1 appartient à un macrocomplexe connectant spatialement des sites de clivages éloignés sur la séquence primaire du pré-ARNr qui seraient rapprochés suite au reploiement de l’ITS2.

Un autre aspect de ce travail de thèse consiste en l’étude de l’assemblage des particules ribonucléoprotéiques et plus spécifiquement du pré-ribosome et des sous-unités ribosomiques eucaryotes. Nous avons utilisé la technique d’immunoprécipitation de chromatine (Ch-IP) pour caractériser l’assemblage d’une structure appelée le « SSU processome ». Celui-ci correspond à un pré-ribosome en formation ainsi que l’assemblage des protéines ribosomiques sur l’ARNr naissant.

Enfin, nous avons étudié le rôle d’une plateforme d’activation de méthyltransférases d’ARN et de protéines, la protéine Trm112 dans la ribogenèse. Nous avons montré que chez la levure, Trm112 est impliquée dans la synthèse du ribosome et dans la progression de la mitose. En absence de cette protéine, les pré-ARNr sont dégradés par un mécanisme de surveillance. Trm112 copurifie avec plusieurs facteurs de synthèse du ribosome dont la méthyltransférase Bud23, impliquée dans la modification post-transcriptionnelle de l’ARNr18S. Trm112 est requise pour cette méthylation et nous postulons que la protéine Bud23 est incapable de se lier aux pré-ribosomes en l’absence de Trm112.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.

Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levuriennes Dim1 et Bud23-Trm112, qu’elles sont requises pour la synthèse et la modification de l’ARNr mature de la petite sous-unité ribosomique, et qu’elles seraient impliquées dans un mécanisme conservé contrôlant la qualité de la voie de biosynthèse du ribosome.

La totalité des FA que nous avons identifiés en cellule humaine sont à la disposition de la communauté scientifique dans une base de données en ligne accessible sur la page www.RibosomeSynthesis.com. Nous espérons que cette ressource contribuera à une meilleure compréhension des mécanismes moléculaires sous-jacents au développement des ribosomopathies et à l’élaboration d’agents thérapeutiques efficaces.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

Après un cycle de la production des protéines, les sous-unités des ribosomes terminés sont dissociés, afin de les rendre disponibles pour de nouveaux cycles de traduction. Si lors de la traduction le ribosome pause, il ne pourra pas terminer la traduction et être recyclé par la voie classique. Un mécanisme de recyclage alternatif a évolué pour dissocier de tels ribosomes arrêtés. Un complexe composé des facteurs Dom34 et Hbs1 induit leur dissociation. Ce complexe est aussi impliqué dans des voies de contrôle qualité qui ciblent des ARN qui causent des arrêts ribosomiques. Dans cette thèse, l'importance de plusieurs sites fonctionnels du complexe Dom34-Hbs1 pour ces voies contrôle qualité des ARNs est étudiée. De plus, la relation entre ces voies et leurs détails sont examiné. Finalement, un nouveau rôle de Dom34-Hbs1, en dissociant des ribosomes inactifs ce qui rend leurs sous-unités disponibles pour de nouveaux cycles de la traduction, est décrit
Protein production is a cyclic process that consists of four stages: initiation, elongation, termination and recycling. During recycling the subunits of terminated ribosomes are dissociated, to make them available for new rounds of translation. If ribosomes stall during translation, ribosomes cannot terminate properly and canonical recycling cannot occur. Cells have mechanisms to rescue these stalled ribosomes. A complex formed by the factors Dom34 and Hbs1 induces their dissociation. This compex in RNA quality control, targeting RNAs that cause ribosomal stalling. In this thesis the importance of several functional sites of the Dom34-Hbs1 complex for the degradation of these RNA sis investigated. Details of and therelationship between RNA quality control pathways in which the complex functions are further investigated. Finally, a new role of this complex, dissociating inactive ribosomes and there by making their subunits available to re-enter the translation cycle is described

L'analyse de la repartition des transcrits de l'arnr16s de plastes d'epinard a permis d'observer une accumulation preferentielle de ces transcrits dans les cotyledons (organes photosynthetiques) par rapport aux racines (organes non photosynthetiques). Cette expression variable suivant les organes de la plante a pu etre associee a la presence de transcrits alternatifs inities au niveau des sites d'initiation de transcription pc et parnt-16s. Alors que parnt-16s est constitutivement represente, le transcrit pc est associe aux organes photosynthetiques. L'expression de l'arnr16s au niveau du site pc ne demarre qu'au 5#e#m#e jours de germination, juste avant l'apparition des cotyledons. Le site d'initiation de transcription pc est localise entre deux sites promoteurs p1 et p2 qui presentent de fortes homolgies avec les promoteurs procaryotiques. L'arn polymerase de e. Coli initie correctement la transcription in vitro au niveau de ces 2 sites. In vivo, seul le site pc est utilise ; p1 et p2 ne sont pas utilises en depit de l'existence de l'arn polymerase chloroplastique de type e. Coli. Dans la deuxieme partie de ce travail, nous avons donc etudie les mecanismes regulant l'expression du 16s au niveau du site pc. Deux complexes nucleoproteiques cl et cs se formant entre la region promotrice du 16s et des proteines chloroplastiques ont ete identifies. Le complexe cs resulte de la fixation des proteines cdf2 (baeza et coll. , 1991). Le complexe cl resulte de la fixation concomitante de l'arn polymerase plastidiale de type e. Coli et des proteines cdf2 sur le promoteur de l'arnr16s. Complexee aux proteines cdf2, cette polymerase ne transcrit pas le 16s au niveau des promoteurs p1 et p2. La transcription de l'arnr 16s au niveau du site pc serait probablement assuree par la polymerase plastidiale monomerique d'origine nucleaire. Le role probable de facteurs d'initiation ou d'activateurs de transcription par cette polymerase, pour les proteines cdf2 est discute

Em eucariotos, a formação das subunidades ribossomais envolve múltiplos fatores, responsáveis pelas etapas de maturação dos rRNAs e por sua associação a proteínas ribossomais. A via de processamento de pré-rRNA é bastante complexa e inclui várias etapas de modificação de nucleotídeos e clivagens endo- e exonucleolíticas. As modificações de nucleotídeos são dirigidas por snoRNPs, formados por snoRNAs e proteínas, que são divididos em duas classes gerais, de box H/ACA (pseudouridilação) e de box C/D (metilação). Dentre os snoRNP de box C/D está o U3, que embora apresente as seqüências características e se associe a proteínas desse grupo de snoRNPs, não dirige metilações no rRNA, mas sim as clivagens iniciais no pré-rRNA 35S. O snoRNA U3 de Saccharomyces cerevisiae é codificado por dois genes que contêm introns, snR17A e snR17B. Embora a via de montagem do snoRNP U3 ainda não tenha sido determinada com precisão, sabe-se que algumas proteínas do core de box C/D ligam-se ao pré-snoRNA U3 co-transcricionalmente, afetando o splicing e o processamento da extremidade 3´ deste snoRNA. A proteína Cwc24p, cuja caracterização funcional foi o objetivo deste trabalho, foi isolada em nosso laboratório interagindo com Nop17p, um fator de montagem dos snoRNPs de box C/D. Cwc24p possui um domínio RING conservado e foi isolada previamente em um complexo contendo o fator de splicing Cef1p. Os resultados aqui obtidos mostram que, de maneira condizente com os dados de interação, Cwc24p é uma proteína nuclear e sua depleção leva ao acúmulo do pré-snoRNA U3, o que acarreta uma diminuição da velocidade de processamento do pré-rRNA 35S. O modelo aqui proposto prevê o recrutamento de Cwc24p para o pré-snoRNA U3 por Nop17p, onde atua como um fator de eficiência do splicing. Estes resultados levaram à conclusão de que Cwc24p está envolvida no splicing do pré-snoRNA U3, afetando indiretamente o processamento do pré-rRNA.
In eukaryotes, the ribosome biogenesis involves a large number of factors, that are responsible for the rRNAs maturation and for their association with ribosomal proteins. The pre-rRNA processing pathway is very complex and includes many steps of nucleotide modifications and endo- and exonucleolytic cleavage reactions. The nucleotide modifications are directed by snoRNPs that are formed by snoRNAs and proteins, divided in two major groups, of box H/ACA (which direct pseudouridilation), or of box C/D (methylation). Although the snoRNP U3 presents the snoRNA sequences and the proteins characteristics of box C/D class, it is not involved in methylation, but rather in the early cleavages of pre-rRNA 35S. U3 snoRNA is transcribed from two intron-containing genes in yeast, snR17A and snR17B. Although the assembly of the U3 snoRNP has not been precisely determined, at least some of the core box C/D proteins are known to bind pre-U3 cotranscriptionally, thereby affecting splicing and 3\'-end processing of this snoRNA. We identified the interaction between the box C/D assembly factor Nop17p and Cwc24p, a novel yeast RING-finger protein which had been previously isolated in a complex with the splicing factor Cef1p. Here we show that, consistently with the protein interaction data, Cwc24p localizes to the cell nucleus, and its depletion leads to the accumulation of both U3 pre-snoRNAs. U3 snoRNA is involved in the early cleavages of 35S pre-rRNA, and the defective splicing of pre-U3 detected in cells depleted of Cwc24p causes the accumulation of the 35S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre-U3 snoRNA splicing, indirectly affecting pre-rRNA processing.

Les ribosomes sont des complexes nucléoproétiques responsables de la traduction. Chez les eucaryotes, la biogenèse du ribosome est un processus complexe très régulé qui fait intervenir un nombre important de facteurs d’assemblages (~200). La construction d’un ribosome est initiée dans le nucléole puis continue dans le nucléoplasme et se termine dans le cytoplasme. La maturation cytoplasmique de la petite sous-unité ribosomale implique la dissociation séquentielle des facteurs d’assemblage tardifs et la maturation finale de l’ARNr 18S. Ce processus est catalysé par l’endonucléase Nob1 qui assure la coupure de l’extrémité 3’ du précurseur de l’ARNr 18S (pré-18S) aboutissant à sa forme mature. Ce mécanisme est coordonné par la protéine Pno1 qui est le partenaire de Nob1. Des informations détaillées sur l’architecture des particules pré-ribosomiques nous ont permis de mieux comprendre les différents intermédiaires de la biogenèse. Cependant, certains aspects fonctionnels comme la conformation adoptée par Nob1 pour assurer la coupure du site D du pre-18S reste encore flou. L’objectif de mon travail a été de mieux comprendre les aspects très tardifs de la maturation cytoplasmique du ribosome. Pour ce faire, nous avons redéfini l’organisation modulaire de l’endonucléase Nob1 chez les eucaryotes pour ensuite étudier son mode d’interaction avec son partenaire Pno1. Des tests fonctionnels in vitro ont été effectués pour étudier le rôle de Pno1 dans la régulation de la coupure par Nob1.Nos résultats nous ont permis de montrer que le domaine catalytique de Nob1 adopte une conformation atypique. En effet le domaine PIN est composé de deux fragments (res 1-104 and 230-255) séparé par une boucle interne qui est importante pour la reconnaissance avec son partenaire Pno1. Nos études nous ont également montré que Pno1 inhibe l’activité de Nob1 probablement en reconnaissant directement l’ARNr substrat, masquant ainsi le site de coupure de l’endonucléase. Ces résultats sont complémentaires et cohérents avec les données structurales de cryo-EM de la particule pré-40S humaine récemment publiées. En effet, Nob1 est dans une conformation incapable de couper le pré-ARNr puisque son domaine catalytique se retrouve à une distance d’environ 30Å de son ARN substrat. Ce phénomène implique donc des changements de conformations ou encore la nécessité de protéine accessoire pour déplacer certains facteurs. La protéine Cinap est impliqué dans la maturation de l’ARNr 18S. Nos études d’interaction avec les protéines localisées au niveau de la plateforme (à savoir RPS14, RPS26, le complexe Nob1/Pno1) ont permis de montrer que Cinap pouvait former un complexe tripartite avec l’endonucléase Nob1 et son partenaire Pno1. De plus, Cinap est capable de reconnaitre RPS26 dans un complexe RPS14-dépendant. Il est important de noter que RPS26 est un composant de la petite sous-unité qui remplace Pno1 dans le ribosome mature. De ce fait le recrutement de RPS26 au sein du pré-ribosome nécessite la dissociation de Pno1 et cet échange serait assurée par Cinap. Sur la base des travaux effectués, nous pouvons proposer un modèle de maturation où la formation du complexe Cinap/Pno1 induirait un changement de conformation permettant à Nob1 de reconnaitre son substrat et donc de catalyser la coupure du site D qui aboutit à la maturation de l’ARNr 18S et donc à la production de la sous-unité 40S mature
Ribosomes are translational machineries universally responsible of protein synthesis. In eukaryote, ribosome assembly is a complex and highly regulated process that requires coordinated action of more than 200 biogenesis factors. Ribosome assembly is initiated in the nucleolus, continues in the nucleoplasm and terminates in the cytoplasm. The cytoplasmic maturation events of the small ribosomal subunit are associated with sequential release of the late assembly factors and concomitant maturation of the pre-rRNA. During final maturation of the small subunit, the pre-18S rRNA is cleaved off by the endonuclease Nob1, which activity is coordinated by its binding partner Pno1. Detailed information on pre-ribosomal particle architectures have been provided by structural snapshots of maturation events. However, key functional aspects such as the architecture required for pre-rRNA cleavage have remained elusive. In order to better understand these late steps of cytoplasmic pre-40S maturation, we first redefine the domain organization of Nob1, then study its binding mode with Pno1 using different tools such as sequence analysis, structure prediction and biochemical experiments and, we then performed functional assay to elucidate the role played by Pno1 during the pre-18S rRNA maturation.Our results have shown that eukaryotic Nob1 adopts an atypical PIN domain conformation: two fragments (res 1-104 and 230-255) separated by an internal loop, which is essential for Pno1 recognition. We also found out that Pno1 inhibits Nob1 activity likely by masking the cleavage site. Our findings further support the recently published cryo-EM structure of the pre-40S, where Nob1 displays an inactive conformation. Moreover, 18S rRNA 3’-end cleavage has to happen and this implies structural rearrangement or requirement of some accessory proteins such as Cinap, an atypical kinase involved in pre-18S processing. Studying the interplay between proteins localized in the pre-40S platform (RPS14, RPS26, Nob1/Pno1 complex) has shown that Cinap is able to form a trimeric complex with Nob1 and its binding partner Pno1. Furthermore, Cinap can recognize RPS26 in a RPS14-dependent manner, which had already been studied with its yeast counterpart. It is important to note that RPS26 is the ribosomal protein replacing Pno1 in the mature ribosome. Our finding clearly suggests a mechanism where RPS26 recruitment to the ribosome requires Pno1 dissociation. This exchange would be carried out by Cinap. Therefore, we can suggest a simplified model as follow: upon binding with Pno1, the newly formed complex (Cinap/Pno1) will trigger a conformational change, which will allow the endonuclease Nob1 to reach its substrate (D-site) and perform its cleavage resulting in mature 18 rRNA generation

Nop53 e uma protena nucleolar, conservada evolutivamente e essencial na levedura Saccharomyces cerevisiae para a biogênese da subunidade maior do ribossomo, 60S. O principal fenotipo causado pela repressão da expressão de Nop53 e o acumulo do intermedi ario de processamento de pre-Rrna, 7S, que tambem e substrato do complexo exossomo na formação do rRNA maduro 5:8S. Nop53 interage diretamente com a subunidade do exossomo Rrp6 e com a subunidade Mtr4 do co-ativador do exossomo TRAMP. O objetivo principal deste trabalho foi o de analisar como a interação entre Nop53 e o exossomo pode modular a atividade deste ultimo. Para isso, foram utilizados metodos bioqumicos, geneticos e de biologia molecular. Os resultados mostrados aqui demonstram que a depleção de Nop53 faz com que mais protenas ribossomais, principalmente da subunidade maior, sejam co-imunoprecipitadas com o core do exossomo, sugerindo que Nop53 possa ter um papel na liberação do exossomo da subunidade pre-60S depois da formação do rRNA maduro 5:8S. Esta hipotese foi conrmada atraves da separação de complexos por centrifugação em gradiente de glicerol, que mostrou a presenca de subunidades do exossomo em complexos maiores na ausência de Nop53, provavelmente correspondendo a partculas pre-ribossomais. Co-imunoprecipitação de RNA com o exossomo na ausência de Nop53 tambem conrmou uma maior associação deste complexo com o pre-rRNA 7S. Como tambem mostrado aqui, alem de interagir com Rrp6, Nop53 interage com subunidades do core do exossomo e a superexpressão de uma destas subunidades, Rrp43, complementa parcialmente a ausência de Nop53 na celula. Estes resultados levaram a conclusão de que Nop53 pode recrutar o exossomo para a partcula ribossomal pre-60S para a maturação do pre-rRNA 7S a 5:8S, e atue tambem na liberação do exossomo, possivelmente atraves de sua interação com a helicase Mtr4.
Abstract Nop53 is a nucleolar, conserved and essential protein in the yeast Saccharomyces cerevisiae, involved in the biogenesis of the large ribosomal subunit 60S. The main phenotype of the depletion of Nop53 in yeast cells is the accumulation of the prerRNA processing intermediate 7S, which is also the substrate of the exosome complex for the formation of the mature rRNA 5:8S. Nop53 directly interacts with the exosome subunit Rrp6, and with the subunit Mtr4 of the TRAMP complex, an exosome co-activator. The main objective of this work was the analysis of the interaction between Nop53 and the exosome and the identication of the mechanism through which Nop53 regulates the exosome activity. The results shown here demonstrate that the depletion of Nop53 leads to a more stable association of the exosome with the pre-60S ribosome particle, as determined by co-immunoprecipitation of proteins with one of the exosome core subunits, and by fractionation of complexes through glycerol gradients. These results suggested that Nop53 could play a role in the release of the exosome after the formation of the mature rRNA 5:8S. This hypothesis was conrmed through the co-immunoprecipitation of pre-rRNA 7S with the exosome in the absence of Nop53. In addition to the interaction with the exosome subunit Rrp6, as shown here, Nop53 also interacts with core subunits of the complex. Interestingly, overexpression of one of these subunits, Rrp43, partially complements the depletion of Nop53. These results led to the conclusion that Nop53 may recruit the exosome to the pre-60S particle for the maturation of the pre-rRNA 7S to the mature 5:8S, but Nop53 may also be involved in the release of the exosome, possibly through its interaction with the helicase Mtr4.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.
CSIR

CSIR
Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.

We describe the use of a tensor mode-1 higher-order singular value decomposition (HOSVD) in the analyses of alignments of 16S and 23S ribosomal RNA (rRNA) sequences, each encoded in a cuboid of frequencies of nucleotides across positions and organisms. This mode-1 HOSVD separates the data cuboids into combinations of patterns of nucleotide frequency variation across the positions and organisms, i.e., "eigenorganisms"' and corresponding nucleotide-specific segments of "eigenpositions," respectively, independent of a-priori knowledge of the taxonomic groups and their relationships, or the rRNA structures. We show that this mode-1 HOSVD provides a mathematical framework for modeling the sequence alignments where the mathematical variables, i.e., the significant eigenpositions and eigenorganisms, are consistent with current biological understanding of the 16S and 23S rRNAs. First, the significant eigenpositions identify multiple relations of similarity and dissimilarity among the taxonomic groups, some known and some previously unknown. Second, the corresponding eigenorganisms identify positions of nucleotides exclusively conserved within the corresponding taxonomic groups, but not among them, that map out entire substructures inserted or deleted within one taxonomic group relative to another. These positions are also enriched in adenosines that are unpaired in the rRNA secondary structure, the majority of which participate in tertiary structure interactions, and some also map to the same substructures. This demonstrates that an organism's evolutionary pathway is correlated and possibly also causally coordinated with insertions or deletions of entire rRNA substructures and unpaired adenosines, i.e., structural motifs which are involved in rRNA folding and function. Third, this mode-1 HOSVD reveals two previously unknown subgenic relationships of convergence and divergence between the Archaea and Microsporidia, that might correspond to two evolutionary pathways, in both the 16S and 23S rRNA alignments. This demonstrates that even on the level of a single rRNA molecule, an organism's evolutionary pathway is composed of different types of changes in structure in reaction to multiple concurrent evolutionary forces.
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DNA- and RNA-binding proteins play a central role in gene regulation, which includes transcriptional control, alternative splicing, post-translational and transcriptional modifications like methylation and acetylation among other roles. In this way, they control most of the working machinery of the cell in direct or indirect manner. Although more than 60 years ago the structure of DNA was proposed by Watson and Crick, our understanding of how RNA- and DNA-binding proteins interact with the genome and transcriptome remains scarce. One of the most important questions in biology is how a large number of DNA- and RNA-binding proteins find their target, interact and later disassociate. These nucleic acid binding proteins either recognizes the unique structural and chemical signatures of the bases (base readout) which give the specificity or it recognizes a sequence-dependent shape (shape readout). Methyltransferases are enzymes with diverse folds, which perform methyltransfer to various substrates using mainly S-adenosyl-L-methionine (AdoMet) as a methyl donor. RNA methylation is one of the most crucial post-transcriptional modifications which influences a wide variety of cellular processes like metabolic stabilization of RNA, quality control in protein synthesis, resistance to antibiotics, mRNA reading frame maintenance, splicing, viral nucleoprotein stabilization among others. Specificity in recognition and methylation in ribosomal RNA (rRNA) methyltransferases is very crucial, as rRNA is highly conserved and lack of specificity would influence the stabilization of RNA and thus, will affect the ribosome. In recent years, rRNA modifications which confer resistance to ribosomal antibiotics have also been observed. The mechanism of recognition to their unique rRNA target site with high selectivity and their evolution still remains an enigma. Thus, the evolution of antibiotic resistance-conferring methyltransferases in pathogenic organisms needs to be investigated from the structural and evolutionary perspective. In the last two decades, many global regulators in both eukaryotes and prokaryotes have been discovered, which promiscuously bind to a large number of DNA sequences. In prokaryotes, they are called as ‘Nucleoid-associated proteins’ (NAPs), which influence the transcriptional process and exhibit multi-specificity or promiscuity. They also take part in the formation of many multi-protein complexes. HU and Integration Host Factor (IHF) are NAPs which belong to prokaryotic DNA-bending protein family (DNABII family). HU and IHF play crucial architectural roles in bacterial DNA condensation and additionally play a regulatory role in many cellular processes. Although sharing structural similarity, the DNA binding and bending features of HU and IHF are strikingly different, allowing them to selectively regulate genes from different genomic locations. HU binds to DNA in a sequence promiscuous manner while IHF is moderately sequence specific. The molecular mechanism of DNA binding multi-specificity (differential specificity with varied binding affinity) of HU/IHF proteins remains unexplored, as little attention has been paid to the determinants at the sequence level. Now, the fundamental question which the author attempted to understand is the structural and evolutionary determinants of specificity in DNA- and RNA-binding proteins. The candidate has taken nucleoid-associated protein HU and SPOUT superfamily RNA methyltransferase as model systems. As the very limited number of structural folds makes up the DNA- and RNA-binding proteins, it is intriguing to examine closely related nucleic acid binding domains or folds carrying out specific functions. Also, we observed that some proteins having a particular structural fold (or homologous ancestry) bind to DNA or RNA with high specificity, while its other homolog binds promiscuously. These observations tempted us to find the sequence and structural determinants which guide this phenomenon, not just specific to only a single protein family, but, determinants are of more general nature, where results can possibly be applied to other nucleic acid binding proteins too. The first part of the thesis reports the crystal structures of native and AdoMet bound ribosomal RNA Methyltransferase from Sinorhizobium meliloti (smMtase), by single anomalous dispersion (SAD) phasing on seleno-methionine substituted crystal, which diffracted to 2.28Å and 2.9 Å resolutions respectively in space group P212121. smMtase belong to an rRNA binding SPOUT superfamily protein, which is fused with an RNA binding L30e domain at the N-terminus. We focused our study on these types of proteins among the large superfamily (henceforth termed as SPOUTL30). The author also has conducted a phylogenetic study, which revealed 11 major clades, out of which we focused our present study in understanding the sequence conservation and variations of 5 (A-E) clades, for which structural, biochemical and functional data is available. These proteins share homology to antibiotic resistance conferring methyltransferases. The availability of experimentally determined structures of native and AdoMet bound smMtase along with an analysis of other homologous crystal structures has enabled a critical examination of factors influencing RNA binding specificity. Also, the thesis reports for the first time an evolutionary and structural inter-connectivity of the three conserved motifs (I-III) in SPOUT superfamily, which is responsible for AdoMet binding and catalysis. The results highlight that both the location of conserved positive and negatively charged residues influence the RNA binding, specificity, and affinity. The conservation of these residues could be at superfamily, family or at clade level, and the position of these charged residues at specific sites, alters their salt-bridge geometry, which ultimately fixes the conformation of RNA-binding residues, thus defining a particular binding site specific to its cognate RNA. The study conducted by the author reveals that the dynamics of salt-bridge and other directional interactions like hydrogen bonding and aromatic interactions essentially determines the specificity of SPOUTL30. The second part of the thesis reports evolutionary, structural and functional studies on nucleoid-associated proteins HU and IHF. To understand the sequence determinants, which influence the degree of DNA binding specificity, we undertook a phylogenetic study in conjunction with analysis of three-dimensional structures. The phylogenetic analysis revealed three major clades, belonging to HU, IHFα, and IHFβ like proteins with reference to E. coli. The author observed statistically significant amino acid compositional bias in the DNA binding sites of HU and IHF clade proteins. The author proposes that the molecular mechanisms giving rise to specificity or multi-specificity depend on a combination effect of the amino acid composition of the binding site, its flexibility, ionic and steric constraints. In continuation of this part of the thesis, the candidate examined the role of protein interacting interface of HU-IHF family proteins, understanding its evolutionary history and utilizing it in designing inhibitors for Mycobacterium tuberculosis HU (MtbHU). The present results give a model example of an evolutionary study of a protein interface of nucleoid-associated protein, which is used to understand the interface and computationally design inhibitors targeting it. The author was a part of the study (Bhowmick et al. 2014, Nature communications) which has determined the crystal structure of Mycobacterium tuberculosis HU, inhibited it using stilbene derivatives (SD1 and SD4) which curtailed the Mtb cell growth. In the present thesis, the candidate observed from microarray analysis that the SD1 stimulon consists of genes involved majorly in lipid biosynthesis pathway, ribosomal genes which affect the overall translation, aerobic respiration pathways, antigenic membrane proteins involved in pathogenicity. Nearly half of the genes in affected by SD1 are essential in nature, thus could explain the curtailing of cellular growth. The whole study provides a system inspired view of probing as well, inhibiting global regulator HU using novel chemical molecules.

Synthetic oligomers of nucleic acids, also called oligonucleotides, are short (typically a few to several nucleotides) nucleic acid strands of potential therapeutic and diagnostic applications. First studies describing the interactions of synthetic oligonucleotides with the RNA of living organisms were presented at the end of the eighties of the twentieth century. Currently, the most commonly used oligomers are modifications of natural nucleic acids that exhibit increased stability in a cellular environment and enhanced stability of the complexes formed with RNA and DNA. This thesis describes the studies of the interactions of 2'-O-methylated RNA and peptide nucleic acid (PNA) oligomers with a functionally important fragment of ribosomal RNA (rRNA). The investigated rRNA region is the so called ribosomal A-site, which serves key functions in the process of translation. This region is mainly responsible for the compliance of the sequences of polypeptide chains synthesised in ribosomes with the information contained in the mRNA template. The research presented in the thesis includes a comparison of the prokaryotic and eukaryotic ribosomal A-site models as well as characteristics of the interactions of 2'-Omethylated RNA and PNA oligonucleotides with rRNA. The studies were performed with the use of molecular dynamics simulations and experimental methods: absorption and fluorescence spectroscopy, isothermal titration calorimetry and gel electrophoresis. Initial studies employing molecular dynamics revealed the lack of appropriate tools for the analysis of nucleic acids simulations. Therefore, an automatic tool for analysing threedimensional structures of RNA and DNA molecules, their full-atom molecular dynamics trajectories or other conformation sets, was created. The presented work is a part of a bigger project, which aims to design effective inhibitors of bacterial proteins translation, based on the synthetic oligonucleotides. The thesis consists of five parts. In the first part, the structure and properties of 2'- O-methylated RNA and PNA oligomers are discussed as well as biological aspects of the study. The second part describes the employed computational and experimental methods. In the third part a novel tool, for the analysis of molecular dynamics trajectories of nucleic acids “MINT” (Motif Identifier for Nucleic Acids Trajectory), is presented. The fourth part describes the results of the computational and experimental studies of the prokaryotic and eukaryotic rRNA models and the interactions of the 2'-O-methylated RNA oligonucleotides with these models. The fifth part is dedicated to the investigation of the potential use of molecular dynamics simulations for prediction of the structural properties and thermal stability of PNA-RNA complexes.
Syntetyczne oligomery kwasów nukleinowych, inaczej oligonukleotydy, to krótkie (zazwyczaj od kilku do kilkudziesięciu nukleotydów) nici kwasów nukleinowych o potencjalnych zastosowaniach terapeutycznych i diagnostycznych. Początek badań nad oddziaływaniami syntetycznych oligonukleotydów z RNA żywych organizmów przypada na koniec lat osiemdziesiątych XX wieku. Obecnie, najczęściej wykorzystywane są oligomery będące modyfikacjami naturalnych kwasów nukleinowych, które cechują się podwyższoną stabilnością w środowisku komórkowym i większą stabilnością tworzonych kompleksów. W przedstawionej pracy badane były oddziaływania oligomerów 2'-O-metylowanego RNA i peptydowego kwasu nukleinowego (PNA) z ważnym funkcjonalnie fragmentem rybosomowego RNA (rRNA) - tzw. miejscem A, które pełni kluczowe funkcje w procesie translacji. Odpowiada ono m.in. za zgodność sekwencji powstających w rybosomie łańcuchów polipeptydowych białek z informacją zawartą w matrycy mRNA. Opisane w pracy badania objęły porównanie właściwości modeli prokariotycznego i eukariotycznego rybosomalnego miejsca A oraz scharakteryzowanie oddziaływań oligonukleotydów 2'-O-metylowanego RNA i PNA z modelowymi fragmentami rRNA. W badaniach wykorzystane zostały obliczenia dynamiki molekularnej, oraz metody eksperymentalne: spektroskopia absorpcyjna i fluorescencyjna, izotermiczne miareczkowanie kalorymetryczne i elektroforeza żelowa. Wstępne badania z wykorzystaniem dynamiki molekularnej ujawniły brak odpowiednich narzędzi do analizy symulacji struktur kwasów nukleinowych. W związku z tym, stworzony został program do analizy struktur kwasów nukleinowych pod kątem charakterystycznych dla nich oddziaływań krótko- i dalekozasięgowych występujących między nukleotydami. Przeprowadzone w ramach pracy badania to część szerszego projektu, którego celem jest stworzenie nowych związków terapeutycznych, opartych o syntetyczne oligonukleotydy. Związki te, wiążąc się z wysoką specyficznością do funkcjonalnych miejsc rybosomów będą zapobiegały syntezie białek bakteryjnych. Przedstawiana praca składa się z pięciu części. W części pierwszej przedstawiono budowę i właściwości 2'-O-metylowanego RNA oraz PNA, oraz omówiono szerzej biologiczny aspekt pracy. Druga część stanowi opis wykorzystywanych metod obliczeniowych i doświadczalnych. W części trzeciej przedstawiony został program do analizy trajektorii dynamiki molekularnej kwasów nukleinowych MINT (ang. Motif Identifier for Nucleic Acids Trajectories). W części czwartej opisane zostały wyniki teoretycznych i doświadczalnych badań modeli prokariotycznego i eukariotycznego rRNA oraz oddziaływań oligonukleotydów 2'-O-metylowanego RNA z tymi modelami. Część piąta poświęcona jest badaniom możliwości wykorzystania obliczeń dynamiki molekularnej do przewidywania właściwości strukturalnych i stabilności termicznej kompleksów PNA z RNA.

Several mechanisms have been proposed for the pol I transcript termination in Schizosaccharomyces pombe. Two well known models are “Pause and Release” and “Torpedo”. Each mechanism explains the role of some of the cis- and trans-factors in transcript termination and the eventual maturation of the ribosomal RNA, but neither mechanism can explain all the experimental observations. A recent study has suggested that each of the two mechanisms can terminate the pol I transcription independently but with significantly less efficiency than the presence of both mechanisms. To help clarify the reasons for the discrepancies in these data, in this study the suggested mechanisms were examined further in three areas by using alternative techniques. First, the effect of uracil concentration or selection times on the transformation frequency of alternative 3’external transcribed spacer (3’ETS) constructs were assessed. Consistent with the previous results a construct containing the full 3’ETS showed the higher transformation frequencies compared with a construct containing only the hairpin or only the termination sites. However, results showed neither the uracil concentration nor selection times have a significant effect on the transformation frequency. Second, to further confirm the “pause and release” mechanism, the termination sites identified by S1 nuclease studies were analyzed using ligation-mediated RT-PCR. The 3’ terminus of the mature 25S rRNA was demonstrated readily but, unexpectedly, the 3’termini of the 3’ETS precursor molecules were not detected, possibly because of their specific structure. Finally, the 3’ extended rRNA precursors were studied by semi-quantitative RT-PCR. These appeared not to correspond with past nuclease protection analyses nor did they demonstrate downstream exonuclease function, observations which question our current understanding of Pol I transcript termination.

The 5’ external transcribed spacer (5’ ETS) of pre-ribosomal RNA, although highly variable in size and sequence, has been shown to be critical for the initiation of rRNA processing. This study further examined the 5’ ETS in Schizosaccharomyces pombe with respect to structural elements that underlie rRNA maturation. Initially, the 5’ ETS/18S rRNA junction region was examined by mutational analyses to detect cis-acting elements critical to known cleavage sites. The results indicated that sequence/structure in the junction region does not direct or strongly influence cleavage at the 5’ end of 18S rRNA. Systematic mutations also were used to examine the significance of previously suggested putative ribosomal protein binding sites or U3 snoRNA binding sites as well as other stem-loop sequences of regions IV, V and VI in the 5’ ETS. The results indicated that the putative U3 snoRNA binding sites were less critical than previously anticipated but have identified elements in regions IV and V with significant influence on the production of mature ribosomal RNA. In vitro studies of interactions between these elements and the U3 snoRNA or cellular protein also were initiated. The results of electrophoretic mobility shift assays indicated a strong interaction between region IV and the U3 snoRNA, suggesting that region IV probably contributes to the function of an important structure in the nucleolar precursor particle, which together with region V and probably other hairpins, may act to organize a stable processing domain. In contrast to the previous studies, which suggested as many as six intermediate cleavage sites in the 5’ ETS of S. pombe, re-examination of termini using hybridization and ligation-mediated RT-PCR indicated only two major cleavage sites. In general the 5’ ETS sequence mutants did not seem to influence the rRNA processing profile significantly but could dramatically affect the quantity of the product, an observation that provided further evidence of quality control, which helps ensure that only functional RNA is incorporated into mature ribosomes. Taken together the results illustrated that various sequence/structural elements in the 5’ ETS could influence or be critical for the maturation of rRNA. The results also support the possibility that the precursor molecule is first organized into one or more processing domains that direct the actual maturation processes.
This study was supported by the Natural Sciences and Engineering Research Council of Canada.

Η ΒΥΠ διαθέτει αντίτυπο της διατριβής σε έντυπη μορφή στο βιβλιοστάσιο διδακτορικών διατριβών που βρίσκεται στο ισόγειο του κτιρίου της.
Στα βακτήρια, η μεγάλη ριβοσωματική υπομονάδα αποτελείται από δύο είδη RNA, το 23S και το 5S rRNA, καθώς και 33 πρωτεΐνες. Ο σχηματισμός του πεπτιδικού δεσμού και η απελευθέρωση της πεπτιδικής αλυσίδας επιτελούνται στη μεγάλη υπομονάδα, όπου εδράζεται το καταλυτικό κέντρο της πεπτιδυλοτρανσφεράσης (PTase). Εκτός αυτού, η μεγάλη υπομονάδα περιλαμβάνει το κέντρο προσδέσεως των μεταφραστικών παραγόντων, το οποίο πυροδοτεί τη GTPase δραστηριότητα των G-πρωτεϊνικών παραγόντων, που εμπλέκονται στη μετατόπιση των υποστρωμάτων και άλλες ριβοσωματικές λειτουργίες. Έχει υποτεθεί ότι το 5S rRNA παίζει ουσιώδη ρόλο στη συγκρότηση του κέντρου της PTase και στη μετάδοση σημάτων μεταξύ του καταλυτικού κέντρου και των ριβοσωματικών συστατικών που διεκπεραιώνουν τη μετατόπιση των υποστρωμάτων. Το ιοντικό περιβάλλον φαίνεται να επηρεάζει καθοριστικά τη διαμόρφωση του 5S rRNA. Για παράδειγμα, έχει βρεθεί ότι οι πολυαμίνες δεσμεύονται εκλεκτικά στο 5S rRNA και επηρεάζουν τη δραστικότητα του έναντι του διμεθυλο-θεϊκού, ενός αντιδραστηρίου-ιχνηθέτη της τριτοταγούς δομής του RNA. Αρχικά ελέγξαμε αν υπάρχουν εξειδικευμένες θέσεις πρόσδεσης των πολυαμινών στο 5S rRNA. Στη συνέχεια, με σκοπό να ελέγξουμε αν η πρόσδεση των πολυαμινών επηρεάζει τη λειτουργία του 5S rRNA, 70S ριβοσώματα προγραμματισμένα με πολύ-ουριδυλικό σχηματίσθηκαν από 50S υπομονάδες, ολικά ή εκλεκτικά φωτοσημασμένες με ένα φωτοδραστικό ανάλογο της σπερμίνης και από 30S ακατέργαστες υπομονάδες. Αυτά τα ριβοσώματα είχαν την ικανότητα να δεσμεύουν AcPhe-tRNA ελαφρώς ισχυρότερα, σε σύγκριση με ριβοσώματα συγκροτημένα από φυσικά συστατικά, μη περιέχοντα πολυαμίνες. Το γεγονός αυτό υποδηλώνει ότι η πρόσδεση πολυαμινών στο 5S rRNA επηρεάζει, σε μικρό βαθμό, τη λειτουργία του παράγοντα επιμήκυνσης EF-Tu. Συζευγμένα, όμως, τα εν λόγω ριβοσώματα με tRNAPhe στην Ε-θέση και AcPhe-tRNAστην Ρ-θέση, επέδειξαν ισχυρότερη καταλυτική δραστικότητα και αυξημένη ικανότητα για μετατόπιση των υποστρωμάτων. Τα αποτελέσματα αυτά εισηγούνται σημαντική εμπλοκή των πολυαμινών στο λειτουργικό ρόλο του 5S rRNA κατά την κατάλυση και μετατόπιση των υποστρωμάτων.
In bacteria, the large ribosomal subunit comprises two RNA species, 23S and 5S rRNA, and 33 proteins. Peptide bond formation and peptide release are catalyzed by the large subunit, where the peptidyltransferase (PTase) center is located. In addition to this center, which triggers the GTPase activities of G-protein factors involved in translocation and other ribosomal functions. It has been hypothesized that 5S rRNA plays essential role in assembling the PTase center and mediating signal transmissions between this center and the translocation machinery. Furthermore, the ionic environment seems to affect the conformation of 5S rRNA. For instance, polyamines have been found to bind specifically to 5S rRNA and influence the 5S rRNA reactivity towards dimethyl-sulfate (DMS), a chemical probe of RNA tertiary structure. Initially we examined whether there are specific sites for binding of polyamines. To test whether the binding of polyamines influence the function of 5S rRNA poly(U)-programmed 70S ribosomes were reconstituted from 50S subunits, totally or specifically photolabelled in their 5S rRNA with a photoreactive analogue of spermine, and native 30S subunits. These ribosomes were found to enzymatically bind AcPhe-tRNA better than ribosomes reconstituted from native components. This means, that furnishing 5S rRNA with spermine slightly influences the elongation factor EF-Tu function. However, equipped with tRNAPhe at the A-site and AcPhe-tRNA at the P-site, these ribosomes exhibited higher catalytic activity and enhanced tRNA translocation efficiency. These results suggest an essential impact of polyamines on the functional role of 5S rRNA in catalysis and translocation of translation substrates.