Telomeres are the ends of linear chromosomes which are protected by a multi-protein complex called shelterin. The proper maintenance of telomeres involves strict control over the length of the TTAGGG telomeric repeat sequences. In part, this is achieved through the action of the shelterin complex component TRF1. TRF1 binds to duplex telomere DNA and acts as a suppressor of telomerase-dependent telomere elongation, however the exact mechanism by which it achieves this is currently unknown. Recent observations with a phospho-specific TRF1 antibody indicate that phosphorylated (pT371)TRF1 localizes at Cajal bodies. Cajal bodies are subnuclear organelles with myriad functions, one of which is to recruit the subunits of the telomerase holoenzyme for assembly and the subsequent targeting of the enzyme to telomeres for elongation. The results presented here demonstrate that this association of phosphorylated (pT371)TRF1 to Cajal bodies is highly specific, requiring its DNA binding capability, and occurring only in Cajal bodies which are not actively involved with telomere extension. While the function of this association has not been elucidated, the data are suggestive of a telomerase-related role being played by phosphorylated (pT371)TRF1 at Cajal bodies, possibly related to its function in suppressing telomere elongation. CSB is a multifunction protein which is implicated in transcription-coupled repair (TCR), base excision repair (BER), and control of transcription. Certain mutations and truncations of CSB are known to cause Cockayne syndrome (CS) in humans, an autosomal recessive progeria with devastating consequences. Unlike other progeria, CS patients do not display increased cancer incidence. Despite this fact, CSB is upregulated and in many cancers. In these cells, removal of CSB leads to apoptosis and increased sensitivity to chemotherapeutic drugs which suggests a dependency on CSB. The CSB of some non-CS cancer patients has also been found to be mutated at several recurring SNPs through the CSB gene. Preliminary examination of some of these SNPs suggests that they may invoke a change in the efficiency of TCR repair of UV-induced DNA damage. The results presented here demonstrate that, for the SNPs examined, there is no significant change in the repair of UV damage as assessed by colony survival assays post UV-treatment. While this may rule out an effect on TCR by these cancer-associated SNPs, it is possible that they may have an effect on CSB’s involvement in other vital cellular processes. / Thesis / Master of Science (MSc)

1 ABSTRACT There are many structures, suborganelles and bodies in the eukaryotic cell nucleus. These domains provide the nucleus with many specific functions. Nucleolus is specialized compartment serves to ribosomes assembly, nuclear speckles or Splicing Factors Compartment play an important role in RNA processing and best studied of them, Cajal bodies (CBs), are involved in snRNP maturation. However, non-membrane substructures are not unique for cell nucleus; processing bodies (P bodies) found in the cytoplasm are proposed to be important places in mRNA degradation pathway. This work is a compilation of four projects focused on non-membrane cellular bodies; namely, nuclear CBs and cytoplasmic P bodies. Both CBs and P bodies are dynamic structures that continuously exchange their components with surrounding environment. In addition to a widely accepted role of CBs in snRNP biogenesis, we show that the CB serves as a place where snRNPs are regenerated after each round of splicing. Thus, CBs are important nuclear compartment involved in snRNP recycling. To further characterize tri-snRNP assembly in CBs we applied kinetic experiments combined with mathematical modeling and created a kinetic model of tri- snRNP formation in the CB that determined kinetic parameters of tri-snRNP formation. Moreover, our kinetic...

Les télomères sont raccourcis à chaque cycle de réplication de l’ADN à cause du problème de réplication des extrémités. Leur longueur dicte la capacité proliférative des cellules eucaryotes. Des télomères trop courts, aillant atteints la limite de Hayflick, feront entrer les cellules en sénescence réplicative. Pourtant dans les cellules progénitrices ainsi que 90% des cellules cancéreuses la longueur des télomères est maintenue par un complexe ribonucléoprotéique, la télomérase. L’assemblage de ce complexe ainsi que son recrutement aux télomères son des processus dynamiques sous le contrôle d’une régulation fine. Toutefois, comment cette régulation à un impact sur la dynamique de la télomérase dans le noyau n’est toujours pas complétement compris. Dans ce projet nous avons développé une nouvelle méthode se basant sur le système MS2 permettant d’observer pour la première des particules uniques d’ARN de télomérase (hTR) par microscopie à fluorescence sur cellules cancéreuse humaines vivantes. Le suivi en temps réel de ces particules aux télomères a permis de révéler que la télomérase scanne activement les télomères via des interactions courtes avec la protéine télomérique TPP1. Des interactions plus stables nécessaires à l’allongement des télomères peuvent être formées grâce à l’appariement entre hTR et l’ADN simple brin du télomère sous contrôle du domaine OB-fold de la protéine télomérique POT1. Le suivi de ces particules au cours du cycle cellulaire a révélé que ces interactions ne sont pas restreintes à la phase S, la phase d’élongation des télomères. La mesure de la dynamique de hTR dans les corps de Cajal (CBs) couplé à des expériences de photo-activation ont pu montrer que hTERT joue un rôle dans la sortie de hTR des CBs. Enfin des mesures d’intensités de fluorescence de hTR aux télomères montrent qu’une seule particule est recruté sur un télomère en phase S. / Telomeres are shortened at each DNA replication cycle, due to the end replication problem. Their length dictates the proliferative capacity of eukaryotic cells. Too short telomeres, reaching the Hayflick limit, will lead the cells to replicative senescence. However, in stem cells and 90% of cancer cells telomere length is maintained by a ribonucleoproteic complex named telomerase. The assembly of this complex and its recruitment to telomeres are process under a fine regulation. Yet, how this regulation impacts the nuclear dynamics of telomerase is not fully understood. In this project, we developed a new method based on the MS2 system to image for the first time telomerase RNA (hTR) particles in living human cancer cells by fluorescence microscopy. The real time tracking of these particles at telomeres revealed that telomerase actively scan all telomeres by short interactions with the TPP1 shelterin protein. Long stable interaction needed for telomere elongation can be made by the pairing of hTR template and single stranded telomeric DNA under the control of the OB-fold domains of the shelterin protein POT1. By tracking telomerase particle during the cell cycle, we revealed that telomerase recruitment to telomeres is not restricted to S phase, when telomeres are elongated. By measuring the dynamics of hTR in Cajal bodies (CBs) with photo-activation and photo-bleaching technics we showed that hTERT play a role in the hTR exit from CBS. Finally, fluorescence intensity measures of hTR at telomeres showed the recruitment of only one particle on a telomere.

Los cuerpos nucleares de Cajal (CBs) son estructuras nucleares implicadas en la biogénesis de ribonucleoproteínas nucleares y nucleolares de pequeño tamaño (snRNPs y snoRNPs) requeridas para el procesamiento nuclear de pre-mRNAs y pre-rRNAs, respectivamente. El CB concentra la proteína coilina, un marcador molecular de esta estructura, snRNPs, el factor de supervivencia de las neuronas motoras (SNM) y las proteínas que comparte con el nucleolo Nopp140 y fibrilarina. Los CB son estructuras dependientes de transcripción, pero los mecanismos de ensamblaje molecular de estos cuerpos nucleares son poco conocidos.En este estudio se utilizan métodos de inmunofluorescencia, expresión ectópica de proteínas del CB y métodos bioquímicos para analizar la importancia de dos modificaciones postraduccionales, la metilación de la coilina y la conjugación con SUMO1 del factor SMN para el ensamblaje molecular de los CBs. Se ha utilizado la línea celular MCF7 como un modelo de hipometilación endógena debido al déficit del gen MTAP. La hipometilación de la coilina conduce al desensamblaje de los CBs y a la relocalización nucleolar de la coilina no metilada. Este efecto revierte en células transfectadas que expresan el gen MTAPwt, indicando que el grado de metilación de la coilina marca su destino nuclear.Respecto a la importancia de la sumoilación en el ensamblaje de los CBs, hemos demostrado la existencia de un subtipo de CBs que concentran SUMO1 y la conjugasa de SUMO Ubc9. En neuronas, hemos detectado la presencia de SUMO durante la fase de reformación de CBs, en la respuesta al estrés. Los experimentos de inmunoprecipitación confirman la interacción de SUMO-1 con el factor SMN y demuestran que la lisina K119, portadora de una secuencia consenso de sumoilación, es esencial para la regulación del número de CBs. / Cajal bodies (CBs) are nuclear structures involved in the biogenesis of small nuclear and nucleolar ribonucleoproteins (snRNPs and snoRNPs) required for nuclear processing of pre-mRNAs and pre-rRNAs, respectively. CBs concentrate the protein coilin, a molecular marker of this structure, snRNPs, the survival of motor neurons factor (SMN) and proteins shared with the nucleolus Nopp140 and fibrillarin. CBs are transcription-dependent structures, but the mechanisms of molecular assembly of these structures are poorly understood.In this study we used inmunofluorescence, ectopic expresion of CB proteins and biochemical methods to analyze the importance of two posttranslational modifications, methylation of coilin and conjugation of SMN with SUMO1, for the molecular assembly of CBs. The cell line MCF7 has been used as a model of endogenous hypomethylation due to the lack of MTAP gene. Coilin hypomethylation leads to the disassembly of CBs and nucleolar relocation of unmethylated coilin. This effect reverses in transfected cells expressing the gene MTAPwt, indicating that the degree of methylation of coilin directs its nuclear destination.On the importance of sumoylation in the assembly of CBs, we have demonstrated the existence of a subset of CBs which concentrate SUMO1 and the SUMO1 conjugase Ubc9. In neurons, we detected the presence of SUMO1 during the reformation of CBs in response to stress. Immunoprecipitation experiments confirm the molecular interaction of SUMO1 with SMN and demonstrate that lysine 119, carrying the SMN sumoylation consensus sequence, is essential for regulating the number of CBs.