Melanosome biogenesis is a highly regulated endosomal maturation process wherein structural fibers harbouring immature melanosomes acquires its biosynthetic proteins through the secretory pathway and finally matures into a functional organelle. These processes were shown to be dependent on several cytosolic protein complexes such as AP (adaptor protein)-1, AP-3, BLOC (biogenesis of lysosome-related organelles complex)-1, -2 and -3; in addition to kinesin motor KIF13A and Rab GTPases 7, 32 or 38. Mutations in the subunits of these complexes or Rab38 result into defective melanosome maturation leading to occulocutaneous albinism, a clinical phenotype commonly observed in Hermansky-Pudlak syndrome (HPS). Moreover, molecular function of these complexes in regulating the biogenesis of melanosome is partially known. The delivery of cargo to maturing melanosomal membranes requires fusion machinery that includes Rab GTPases, tethering factors and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. However, the SNAREs involved in the transport of cargo to melanosomes is poorly understood. In this study entitled as “understanding the SNARE dynamics during melanosome biogenesis” we focus on functional role of endosomal Qa-SNARE protein, Syntaxin 13 (formally called STX12, herein referred to as STX13) in the organelle biogenesis and its transport in and out of melanosome. Moreover, these studies show that STX13-mediated cargo transport require a melanosomal membrane localized R-SNARE VAMP7 and these SNAREs are interdependent on each other in regulating their steady state distribution. In addition, this study illustrated the possible mechanism of SNARE recycling which occurs indirectly through AP-3 complex. Thus, these studies underscore the STX13‟s role in cargo transport to maturating melanosomes and its trafficking routes to and from the melanosomes. Chapter-I describes the literature review on melanosome biogenesis; Chapter-II lists the experimental procedures used in this study and Chapter-III to V focuses on results and discussion, segregated into three sections. Chapter-III: Screening and identification of endosomal SNAREs involved in the trafficking of melanosomal proteins. Our preliminary RNAi screen for SNAREs involved in melanosome biogenesis revealed STX13 as one of the Qa-SNARE affecting pigmentation and cargo transport. STX13, a recycling endosomal SNARE has been reported to interact with pallidin, a subunit of BLOC-1; however the functional role of this interaction in pigment formation is unknown. In addition, previous studies from our lab have shown that STX13 colocalize with endosomal Rab11 and partially with EEA1- or Rab5-positive organelles in melanocytes. Together, these observations insinuated us to characterize the functional role of STX13 in melanosome biogenesis. Upon STX13 inactivation, wild type mouse melanocytes showed hypopigmentation due to mistargeting of cargo such as TYRP1 and TYR to lysosomes. Knockdown of STX13 dramatically decrease the population of immature and mature melanosomes. Moreover, STX13 associate with the melanosome cargo on endosomal tubular structures. In addition, deletion of regulatory domain in STX13 increases the cargo transport to melanosomes due to its increased SNARE activity. This is possibly due to loss in intracellular regulation of SNARE occur through multiple factors such as SM (Sec1p/Munc18) proteins. Together this data suggests that STX13 mediates cargo transport to melanosomes from recycling endosomes. Chapter-IV: Functional characterization of the SNAREs involved in melanosomal maturation. Several in vitro studies have shown that a set of four SNAREs such as Qa, Qb, Qc (or Qbc) and R control the membrane fusion event duing the cargo transport. Additionally, this process is further regulated by SM proteins in in vivo. Electron microscopic studies in melanocytes have shown that melanosomal proteins were delivered to the melanosomal membrane through recycling endosomal tubular domains. Moreover, our RNAi screen show that STX13 possibly acts as Qa-SNARE in mediating the fusion events between melanosomal membranes and the endosomal tubular or vesicular intermediates. However, the role of other SNAREs for this membrane transport is unknown. It has been shown that the expression of VAMP family SNAREs such as VAMP3, VAMP7 and VAMP8 increased with melanogenesis upon differentiation of melanoma cells. VAMPs belong to the class of R-SNAREs, in which VAMP7 is known to interact with VARP (abbreviation) and AP-3 (mediates the trafficking of TYR) separately, and these molecules are known to regulate the cargo transport to melanosomes. However, the precise role of VAMP7 in pigment granule maturation is unknown. Therefore, we set out to characterize the functional role of VAMP7 in melanosome biogenesis. VAMP7 has been shown to localizes to multiple sub-cellular compartments and regulate the several transport steps in other cell types. Our study found that GFP-epitope tagged either human or rat VAMP7 localize to melanosomes at steady state in wild type mouse melanocytes. Knockdown of VAMP7 causes hypopigmentation of melanocytes and misroutes the cargo to lysosomes. Further, the inactivation of VAMP7 in melanocytes phenocopies the STX13 depletion, suggesting both the SNAREs are required for the melanosome biogenesis. In addition, knockdown of STX13 target the VAMP7 to lysosomes; while inactivation of VAMP7 affect the localization of STX13 to recycling tubular structures. Subsequently, the dominant active mutants of STX13 were not able to rescue the pigmentation or cargo transport defects in VAMP7 knockdown melanocytes. Together, the data suggests that STX13 functions from recycling endosomes and VAMP7 on melanosome membrane for the transport of cargo to melanosomes Chapter-V: Understanding the mechanism of STX13 recycling during melanosome biogenesis. At steady state, SNAREs are localized to the membranes of specific organelles where they mediate or regulate the membrane fusion. During this process, three or two Q-SNAREs on one membrane (in a trans-SNARE complex, possibly formed by Qa, Qb, Qc or Qbc) interact with a R-SNARE on another member to form a SNAREpin complex. Post-fusion, SNAREs are disassembled by SNAP and NSF proteins and then recycled back to the original compartment for next round of fusion. Here, we address the mechanism of post-fusion recycling of STX13 from melanosomes to endosomes. Previous studies have shown that STX13 mislocalize to melanosomes in AP-3-deficient melanocytes, suggesting a role for AP-3 in recycling the SNARE from melanosomes. Bioinformatic analysis of the N-terminal region of STX13 revealed the presence of two canonical adaptor binding motifs 3YGP6L and KETNE80L81L, resembling the tyrosine-based (YXXø) and dileucine-based motif [DE]XXXL[LI], recognized by several adaptor proteins. Point mutagenesis of these motifs in STX13 had no effect on their steady state distribution indicating that STX13 possibly uses non-canonical residues for its recycling. Further, deletion of the N-terminal region (either 1-129 or 14-129 aa) in STX13 redistributes the SNARE to melanosomes. Moreover, the activity and the trafficking of recycling defective STX13 mutants are dependent on another HPS complex, BLOC-2 and the SNARE, VAMP7. Absence of 1-129 region in STX13 or mutations in the subunits of AP-3 perturbs the steady state localization of STX13 suggesting an indirect role for AP-3 in recycling of STX13 to endosome via non canonical motifs present in its 1-129 aa region.

Dans un premier temps, nous avons observé la chronologie des altérations de la biogenèse et de la fonction mitochondriale dans les ventricules droit (VD) et gauche (VG) et le muscle gastrocnémien (GAS) dans un modèle animal d’hypertension artérielle pulmonaire (HTAP). Nous avons constaté une diminution précoce des facteurs impliqués dans la biogénèse mitochondriale du GAS. Plus tard, les mêmes anomalies apparaissaient dans le VD. Au stade décompensé de l’insuffisance cardiaque droite s’ajoutaient une diminution de la protéine PGC-1 , de l’activité de la citrate-synthase et de la respiration mitochondriale. L’expression des ARNm et la respiration mitochondriale du VG n’étaient pas modifiées de façon significative.Dans un deuxième temps, nous avons étudié l’effet de l’entraînement en mode excentrique sur le même modèle d’HTAP. La survie des rats entraînés n’était pas différente de celle des rats sédentaires et la tolérance hémodynamique évaluée par échocardiographie et cathétérisme cardiaque a été bonne. Le bénéfice de l’entraînement s’est traduit par une augmentation de la vitesse maximale de course dans les deux groupes entraînés, malades et témoins. / We assessed the time courses of mitochondrial biogenesis factors and respiration in the right ventricle (RV), gastrocnemius (GAS) and left ventricle (LV) in a model of pulmonary-hypertensive (PH) rats induced by monocrotaline (MT). The expression of the studied genes was decreased early in the MT GAS. At 4 weeks, the MT GAS and MT RV showed decreased mRNA levels whatever the stage of disease, but PGC-1 protein and citrate-synthase activity were significantly reduced only atthe decompensated stage. The functional result was a significant fall in mitochondrial respiration at the decompensated stage in the RV and GAS. The mRNA expression and mitochondrial respiration were not significantly modified in the MT LV. Secondly, we assessed the effects of eccentric exercise training (ECCt) in MT rats with PH. ECCt was initiated 2 weeks after MT injection for 4 weeks. The trained MT rats survival was not different from that of sedentary rats. ECCt was not detrimental on hemodynamic condition estimated by echocardiography and right heart catheterization. Maximal speed significantly increased in trained rats. The mRNA expression of mitochondrial biogenesis factors were not significantly modified in skeletal muscle and in RV.

Made available in DSpace on 2016-06-02T19:22:12Z (GMT). No. of bitstreams: 1 6669.pdf: 3530464 bytes, checksum: bd826d7ee4a6c0bb3ebc74dc5752604e (MD5) Previous issue date: 2015-04-10 / Financiadora de Estudos e Projetos / The decrease of regulate the estrogen production that occurs at menopause is typically followed by increase of several deleterious changes in the skeletal muscle system. Menopause is mimicked experimentally by a technique called ovariectomy. The ovariectomy produces increased total body mass, changes in body composition and lipid profile, reduction in skeletal muscle (sarcopenia) and bone mineral mass (osteopenia). Mitochondria play a crucial role in a myriad of cellular processes including oxidative phosphorylation, biosynthetic pathways and programming of cell death. Alteration of mitochondrial biogenesis markers in ovariectomized rats and the effects of resistance training (RT) and estrogen replacement (RE) are unclear. Purpose: This study aimed to investigate the effects of Ovariectomy (Ovx), RT and ER on markers of mitochondrial biogenesis and protein expression related to oxidative capacity in the rat gastrocnemius pool. Methods: ER was performed using Silastic® capsules. During the 12-week RT, the animals climbed a ladder with weights attached to their tails. RT began simultaneously for all experimental groups. Gene expression was analysed by RT-PCR, and protein content was determined by western blotting. Results: The estrogen deficiency associated with Ovx decreased the gene expression of the mitochondrial biogenesis markers PGC-1&#945; (~73%), NRF-1 (~44%), and TFAM (~53%) (p<0.05) and decreased the protein expression of phosphorylated AMPK, CREB and AKT, which are related to oxidative capacity, compared to the Sham-Sed group. RT increased PGC- 1&#945; (~59%) and TFAM (~48%) expression compared to the Ovx-Sed group. The combination of RT and ER was superior to the Ovx-Sed and Ovx-RT treatments regarding the gastrocnemius muscle. Conclusions: This study showed that ovaries removal affects transcription factors that regulate mitochondrial biogenesis in skeletal muscle. According to our results and evidence from the literature, and estradiol levels of exercise appear to play an important role in the protection of mitochondrial dysfunction in skeletal muscle of rats. / Introdução: A diminuição da produção regular do estrógeno que ocorre na menopausa é tipicamente seguida pelo aumento de várias alterações deletérias no sistema musculoesquelético. A menopausa é mimetizada experimentalmente por uma técnica chamada ovariectomia. A ovariectomia produz aumento da massa corporal total, alterações na composição corporal e no perfil lipídico, redução da musculatura esquelética (sarcopenia) e da massa mineral óssea (osteopenia). As mitocôndrias desempenham papel crucial em uma miríade de processos celulares que incluem fosforilação oxidativa, vias de biossíntese e programação da morte celular. Alteração dos marcadores de biogênese mitocondrial em ratas ovariectomizadas, bem como os efeitos do treinamento resistido e reposição de estrógeno não são totalmente conhecidas. Objetivos: Investigar os efeitos da ovariectomia, do treinamento resistido e da reposição de estrógeno sobre a biogênese mitocondrial e capacidade oxidativa do músculo esquelético de ratas. Materiais e Métodos: A reposição de estrógeno foi realizada com cápsula Silastic. O treinamento resistido consistiu de 12 semanas em que os animais subiram uma escada com pesos atados às suas caudas. A expressão gênica foi analisada por RT-PCR e o conteúdo de proteína por Western Blotting. Resultados: A deficiência de estrogénio, associada à ovariectomia, reduziu a expressão gênica de marcadores de biogênese mitocondriais, tais como PGC-1&#945; (~ 73%), NRF-1 (~ 44%), TFAM (~ 53%) (p <0,05) e o conteúdo de proteína relacionada à capacidade oxidativa, como AMPK, CREB e AKT quando comparado com grupo Sham-Sed. O treinamento resistido aumentou esses marcadores, tais como PGC-1&#945; (~ 59%) e TFAM (~ 48%) em relação ao grupo Ovx-Sed. Conclusões: O presente estudo mostrou que a remoção dos ovários afeta fatores de transcrição que regulam a biogênese mitocondrial no músculo esquelético. De acordo com os nossos resultados e evidências da literatura, os níveis de estradiol e de exercício parecem desempenhar um papel importante na proteção da disfunção mitocondrial no músculo esquelético de ratas.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2016-09-12T20:34:22Z No. of bitstreams: 1 TeseMMD.pdf: 2837994 bytes, checksum: 0f8e9168f951c057cd3d2b3582ba6b01 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T18:42:13Z (GMT) No. of bitstreams: 1 TeseMMD.pdf: 2837994 bytes, checksum: 0f8e9168f951c057cd3d2b3582ba6b01 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T18:42:48Z (GMT) No. of bitstreams: 1 TeseMMD.pdf: 2837994 bytes, checksum: 0f8e9168f951c057cd3d2b3582ba6b01 (MD5) / Made available in DSpace on 2016-09-13T18:42:59Z (GMT). No. of bitstreams: 1 TeseMMD.pdf: 2837994 bytes, checksum: 0f8e9168f951c057cd3d2b3582ba6b01 (MD5) Previous issue date: 2015-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Decreased levels of estrogen are associated with a decline in brain bioenergetics, which could be associated with a decrease in mitochondrial biogenesis (MB). Endurance training (ET) has been shown to increase markers of MB within the brain. However, the molecular events associated with this process were only investigated for ET but not for resistance training (RT) and hormone replacement therapy (HRT). In the study, we attempted to investigate the effects of ovariectomy (Ovx), RT and HRT on markers of MB (mRNA expression of peroxisome proliferator - activated receptor - γ coactivator 1 (PGC - 1α), nuclear respiratory fator- 1 (NRF - 1), and mitochondrial transcription factor A (TFAM)) in specific brain regions, cortex (CX), hippocampus (HC), and hypothalamus (HT) in rats. Sprague - Dawley adult female rats were grouped into six groups: sham - operated sedentary (Sham - Sed); Ovx - Sed; Sham - RT; Ovx - RT; Ovx – Sed - HRT and Ovx - RT - HRT. The animals in HRT groups received subcut aneously implanted silastic capsules with a solution of 180 μg 17β - estradiol/ml sunflower oil. A 12- week RT period, during which the animals climbed a 1.1 - m vertical ladder with weights attached to their tails, was used. The sessions were performed once every 3 days, with 4 - 9 climbs. Gene expression was analyzed by RT - PCR by the ∆∆Ct method. The Ovx decreased the gene expression of molecules related to BM, PGC - 1α (28%), NRF - 1 (29%) and TFAM (20%) in the HC. These Ovx - induced lower gene expressions were totally restored in this structure by RT. RT increased the markers of MB, PGC - 1a (~33%), NRF - 1 (~31%) and TFAM (~44%) in CX, HC and HT. These findings suggest that OVX decreases brain MB, in our data observed in HC, and RT increases brain MB, which may have important implications with respect to various central nervous system diseases and age - related dementia that are often characterized by mitochondrial dysfunction. Stimulation or enhancement of mitochondrial biogenesis may prove a novel neuroprotective strategy in the future. / A diminuição dos níveis dos hormônios ovarianos está associado a um declínio na bioenergética cerebral, o que poderia estar associado a uma diminuição da biogênese mitocondrial (BM). O treinamento de endurance (TE) foi demonstrado aumentar a expressão gênica dos marcadores da BM em diferentes regiões do cérebro. No entanto, os eventos moleculares da BM ainda não foram investigados em outros tipos de treinamento, como, o treinamento resistido (TR) ou em outros tipos de modelo experimental, como a ovariectomia (Ovx) e a terapia de reposição hormonal (TRH). Assim, o objetivo deste trabalho foi analisar os efeitos da Ovx, do TR e da TRH sobre a expressão gênica dos marcadores chaves da biogênese mitocondrial, o coativador 1α do receptor gama ativado por proliferador de peroxissoma (PGC-1α), o fator respiratório nuclear 1 (NRF-1) e o fator de transcrição mitocondrial A (TFAM) em diferentes regiões do cérebro, córtex (CX), hipocampo (HC) e hipotálamo (HT) de ratas. Trinta e seis ratas adultas Sprague - Dawley foram distribuídas em seis grupos experimentais (n = 6 por grupo): (I) sedentário sham-operação (Sed-Sham); (II) Ovx - Sed; (III) Sham-TR; (IV) Ovx - TR; (V) Ovx – Sed - TRH e (VI) Ovx- TR- TRH. Os animais dos grupos Ovx foram submetidos aos procedimentos cirúrgicos de remoção bilateral dos ovários, dos grupos TRH foram submetidos aos procedimentos cirúrgicos de implantação subcutânea de cápsulas silásticas preenchidas com uma solução de 180 μg de 17 β - estradiol/ml de óleo de girassol e os animais dos grupos TR foram submetidos a doze semanas de treinamento resistido progressivo em escada, sendo as sessões de treinos realizadas uma vez a cada 3 dias. A expressão gênica foi analisada por PCR-RT e determinada pelo método do ∆∆Ct. O grupo Ovx - Sed apresentou uma menor expressão gênica dos marcadores chaves da BM no HC quando comparado ao grupo Sham - Sed. A expressão do PGC - 1α foi 28% menor, do NRF - 1 29% menor e do TFAM 20% menor. Estas menores expressões gênicas no HC foram restauradas nas ratas do grupo Ovx - TR e parcialmente restauradas nas ratas do grupo Ovx – Sed - TRH. Adicionalmente, as ratas do grupo Sham-TR apresentaram maior expressão gênica nas três áreas investigadas (CX, HC e HT), PGC - 1α ~33% maior, NRF - 1 ~31% maior e TFAM ~44% maior. Estes resultados sugerem que o declínio na concentração circulante dos hormônios ovarianos diminui a BM no cérebro, em nossos resultados observados no HC, e que o TR pode aumentar a BM no cérebro, o que pode ter importantes implicações no que diz respeito a várias doenças do sistema nervoso central e demência relacionada com a idade que são frequentemente caracterizadas por disfunção mitocondrial. Sendo assim, a estimulação da BM cerebral estimulada pelo exercício pode ser uma importante estratégia de neuroproteção.

Submitted by Livia Mello (liviacmello@yahoo.com.br) on 2016-10-03T14:14:11Z No. of bitstreams: 1 DissCTM.pdf: 1497226 bytes, checksum: e33fe63139fc98a7e56c4936a60ee50c (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-10T19:42:38Z (GMT) No. of bitstreams: 1 DissCTM.pdf: 1497226 bytes, checksum: e33fe63139fc98a7e56c4936a60ee50c (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-10T19:42:49Z (GMT) No. of bitstreams: 1 DissCTM.pdf: 1497226 bytes, checksum: e33fe63139fc98a7e56c4936a60ee50c (MD5) / Made available in DSpace on 2016-10-10T19:42:59Z (GMT). No. of bitstreams: 1 DissCTM.pdf: 1497226 bytes, checksum: e33fe63139fc98a7e56c4936a60ee50c (MD5) Previous issue date: 2016-04-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Menopause, permanent discontinuation phase of ovarian follicular activity, triggers tissue and molecular changes in the body of women. The obesity and sarcopenia are two important deleterious effects, affect the functional independence and therefore the quality of life of the same. Resistance training (RT), caloric restriction (CR) are interventions that can minimize and slow down these degenerative processes related to menopause. The objective of this study was evaluate the effects of TR and RC on the biogenesis markers and mitochondrial dynamics on the quadríceps mucle of ovariectomized rats. Ratas Holtzman were divided into 8 groups (n = 10) Sham sedentary, trained, caloric and association training restriction with caloric restriction (SHAM SED, SHAM TR, SHAM RC AND SHAM TR-RC), ovariectomized sedentary, trained, caloric restriction and association training with caloric restriction (OVX SED, OVX TR, OVX and OVX RC TR-RC). The ovariectomy surgery (OVX) and pseudo-ovariectomy (Sham) was conducted when the rats reached 250g body weight. Two days after surgery, began the control of food intake and after 10 days of recovery from surgery, began the TR which consisted of 13 weeks of climbing training vertical ladder, with 72 hours between training sessions. The intensity of TR was 65, 85, 95 and 100% of the predetermined maximum load for each load proportion. After the fourth climbing, it was added 30g to determine the new maximum filler loading. Euthanasia of the animals after 13 weeks was TR and 48 hours after the last training session. The quadriceps muscle of the right hind foot was excised and stored at -80°C for analysis of gene expression and protein mediators of biogenesis and mitochondrial dynamics. The ovariectomy in rats altered mitochondrial biogenesis and dynamics as well as body weight and food consumption. The proposed interventions have been effective in minimized the body mass gains and reverse the molecular changes found in ovariectomy, especially in relation to mitochondrial biogenesis and dynamics. / A menopausa, fase de interrupção permanente da atividade folicular ovariana, desencadeia alterações teciduais e moleculares no organismo das mulheres. O ganho de massa corporal e a sarcopenia são dois importantes efeitos deletérios que ao longo do tempo, afetam a independência funcional e consequente qualidade de vida das mesmas. O treinamento resistido (TR), a restrição calórica (RC) são intervenções que podem amenizar e retardar esses processos degenerativos relacionados à menopausa. O objetivo deste trabalho foi avaliar os efeitos do TR e RC sobre os marcadores de biogênese e dinâmica mitocondrial sobre o quadríceps de ratas ovariectomizadas. Ratas Holtzman foram alocadas em 8 grupos (n=10) Sham sedentárias, treinadas, restrição calórica e associação treinamento com restrição calórica (SHAM SED, SHAM TR, SHAM RC E SHAM TR-RC), ovariectomizadas sedentárias, treinadas, restrição calórica e associação treinamento com restrição calórica (OVX SED, OVX TR, OVX RC e OVX TR-RC). As cirurgias ovariectomia (OVX) e pseudo-ovariectomia (SHAM) foram realizadas quando as ratas alcançaram 250g de massa corporal. Dois dias após as cirurgias, iniciou-se o controle do consumo alimentar e após 10 dias de recuperação das cirurgias, iniciou o TR que consistiu em 13 semanas de treinamento de escalada em escada vertical, com 72 horas de intervalo entre as sessões de treinamento. A intensidade do TR foi de 65, 85, 95 e 100% da carga máxima de carregamento previamente determinada para cada rata. Após a quarta escalada, acrescentou-se 30g para determinar a nova carga máxima de carregamento. A eutanásia dos animais ocorreu após 13 semanas de TR e 48 horas após a última sessão de treino. O músculo quadríceps da pata traseira direita foi excisado e armazenado a -80°C para as análises da expressão gênica e proteica dos mediadores da biogênese e dinâmica mitocondrial. A ovariectomia em ratas alterou a biogênese e dinâmica mitocondrial, bem como a massa corporal e o consumo alimentar. As intervenções propostas foram eficazes em amenizar os ganhos da massa corporal e reverter as alterações moleculares encontradas na ovariectomia, principalmente em relação à biogênese e dinâmica mitocondrial.

Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...

A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

Mitochondrion is an endosymbiotic organelle synthesizing ~1% of its proteome, while remaining ~99% of the proteins are encoded by the nuclear genome and translated on the cytosolic ribosome. Therefore active mitochondrial biogenesis requires efficient protein transport destined for the different sub-compartments. Mitochondrion contains specialized translocation machineries in the outer and in the inner membrane known as TOM40 and TIM23-complex respectively. Import of a majority of mitochondrial proteome is mediated by inner membrane presequence translocase (TIM23 complex). However, the structural organization of Tim23-complex and mechanisms of mitochondrial inner membrane protein translocation is still elusive. Therefore, the present thesis addresses above elusive questions. Chapter 2 highlights the functional significance of different segments of Tim23 in regulating the conformational dynamics of the presequence-translocase- Tim23 is the central channel forming subunit of the presequence-translocase which recruits additional components for the assembly of the core complex. However the functional significance of different segments of Tim23 was not understood due to the lack of suitable conditional mutants. Our study has reported many conditional mutants from different segments of Tim23 which are precisely defective in the organization of the core complex and in the recruitment of the import motor component which enhances our understanding of protein translocation across mitochondrial inner membrane. Chapter 3 highlights the functional cooperativity among mtHsp70 paralogs and orthologs using Saccharomyces cerevisiae as a model organism- mtHsp70s are implicated in a broad spectrum of functions inside the mitochondria. In case of lower eukaryotes gene duplication event has given rise to multiple copies of Hsp70s thereby presenting an opportunity of division of function among these paralogs. The mitochondria of yeast Saccharomyces cerevisiae contains three Hsp70s, including Ssc1, Ssq1 and Ssc3 (Ecm10). The Ssc1 is essential for protein translocation and de novo protein folding functions while Ssq1 is needed for the Fe/S cluster biogenesis inside the mitochondria. Although it has been proposed earlier that, Ssc1 and Ssc3 possesses overlapping functions in protein translocation as a part of import motor in the Tim23-complex. However the physiological relevance and experimental evidences in favor above hypothesis was not established clearly. Our study has reported Ssc3 as an ‘atypical chaperone’ which cannot perform the generalized chaperone functions due to the conformational plasticity associated with both the domains of Ssc3 resulting into weaker client protein affinity, altered interaction with cochaperones and dysfunctional allosteric interface. Additionally, we have also highlighted the role of Nucleotide-binding domain in determining the functional specificity among Hsp70 paralogs and orthologs.

Chez les eucaryotes, l’information génétique est transcrite en ARN messager qui subit plusieurs étapes de maturation et évènements d’assemblage avant d’être exporté hors du noyau. Ces modifications du transcrit sont effectuées par de nombreux facteurs protéiques recrutés au transcrit naissant, formant ainsi une particule ribonucléoprotéique (mRNP). La biogenèse du mRNP est étroitement liée avec la transcription et le contrôle qualité afin d’assurer l’efficacité et l’exactitude de la production de mRNPs matures. Des études récentes suggèrent que les membres du complexe THO-Sub2 pourraient être des facteurs cruciaux dans le couplage de la transcription, de la biogénèse du mRNP et de l’export. Dans notre groupe, nous avons mis en oeuvre un essai novateur pour étudier la biogénèse du mRNP et le contrôle qualité, basé sur l’expression du facteur Rho bactérien dans Saccharomyces cerevisiae. Rho interfère avec l’assemblage adéquat du mRNP et génère des transcrits aberrants qui sont dégradés par la machinerie de dégradation nucléaire. Dans cette étude, nous avons utilisé le système expérimental Rho pour mieux comprendre Rrp6 et l’implication de l’exosome dans la dégradation des transcrits liée au contrôle qualité, ainsi que pour mieux caractériser le rôle et la fonction du complexe THO-Sub2 dans le processus de biogénèse du mRNP. Les résultats obtenus révèlent une différence intéressante dans le comportement des membres du complexe THO sous l’action de Rho et dévoilent leur dépendance à la liaison à l’ARN, ce qui n’aurait pas pu être observé avec d’autres techniques expérimentales. Cela confirme le potentiel attendu du système expérimental basé sur Rho dans l’étude des facteurs protéiques impliqués dans la biogénèse et le contrôle qualité du mRNP. / In eukaryotes, the genetic information is transcribed into messenger RNA which undergoes various processing and assembly events prior to its export from the nucleus. These transcript modifications are performed by numerous protein factors recruited to the nascent transcript, thus making a messenger ribonucleoprotein particle (mRNP). mRNP biogenesis is tightly interconnected with both transcription and quality control to ensure efficiency and accuracy in production of mature mRNPs. Recent findings suggest that members of THO-Sub2 complex might be crucial factors in coupling transcription, mRNP biogenesis and export. In our group, we have implemented an innovative assay to study mRNP biogenesis and quality control, based on the expression of the bacterial factor Rho in Saccharomyces cerevisiae. Rho interferes with proper mRNP assembly and generates aberrant transcripts degraded by the nuclear degradation machinery. In this study, we use Rho experimental system to expand our findings on Rrp6 and exosome involvement in quality control degradation of transcripts, as well as to better characterize the role and function of THO-Sub2 complex in the process of mRNP biogenesis. Obtained results reveal an interesting difference in behavior of THO complex members upon Rho action and disclose their dependence on binding to the RNA, which could not be observed by other experimental techniques. This substantiates the expected potential of Rho-based experimental system in the study of protein factors involved in mRNP biogenesis and quality control.