No carcinoma papilífero de tiroide (CPT) ocorrem alterações na via MAPK (do inglês Mitogen-Activated Protein Kinase), sendo a mais frequente mutação a BRAFT1799A. A via MAPK é modulada pelo miRNA, como o let-7 que estão pouco expresso no CPT. A biogênese de let-7 é controlada por proteínas ligantes de RNA LIN28 que inibem o processamento das formas primária e precursora de let-7 (pri-let-7 e pre-let-7). Avaliamos a expressão gênica de LIN28, pri-let-7f, pre-let-7f e let-7f por rtPCR em tempo real, utilizando RNA total das linhagens TPC-1 e BCPAP de PTC e amostras tumorais de pacientes com CPT. A linhagem não tumoral Nthyori 3-1 foi utilizada para induzir LIN28. Observa-se redução de let-7f em 4 dos 5 pacientes analisados enquanto que LIN28A está aumentado em 2 pacientes com BRAFT1799A. Nota-se maior expressão de pri-let-7f em TPC-1 e menor expressão de pre-let-7f e let-7f. Observa-se maior expressão de LIN28A em TPC-1, enquanto que LIN28B está mais expresso em BCPAP com BRAFT1799A. A inibição de BRAFV600E em BCPAP diminui LIN28B e aumenta let-7f, enquanto que a indução de LIN28B reduz a expressão de let-7f. Concluímos que há relação inversa entre LIN28 e let-7f no CPT e LIN28 influencia a diminuição de let-7f podendo assim contribuir com tumorigênese tireoidiana. / In papillary thyroid cancer (PTC) changes in MAPK (Mitogen-Activated Protein Kinase) pathway are common being BRAFT1799A mutation the most frequent alteration. MAPK pathway is modulated by miRNA such as let-7, an under-expressed miRNA in PTC. The RNA binding protein LIN28 controls let-7 biogenesis, blocking primary and precursor let-7 (pri-let-7 e pre-let-7) processing. We evaluated LIN28, pri-let-7f, pre-let-7f and let-7f gene expression by real time RTPCR using total RNA of human PTC sample and PTC cell lines TPC-1 and BCPAP. Non-tumoral cell line Nthyori 3-1 was used to evaluate LIN28B influence in let-7f. We observed decreased let-7f expression in 4 out of 5 PTC patients, and increased LIN28A in 2 patients with BRAFT1799A mutation. TPC-1 cells express higher levels of pri-let-7f while pre-let-7f and let-7f are less expressed. We noted that LIN28A is more expressed in TPC-1, while LIN28B is more expressed in BCPAP cells with BRAFT1799A. The inhibition of BRAFV600E in BCPAP decreases LIN28B and increases let-7f, while the induction of LIN28B in Nthyori 3-1 reduces let-7f expression. We conclude there is an inverse association between LIN28 and let-7f in PTC, and that LIN28 influences let-7f reduction which could contribute to thyroid tumorigenesis.

By means of reverse-transcription polymerase chain reaction, overexpression of ZNF146 was detected in two human HCC cell lines HepG2 and Hep3B and a clear relationship between HCC and overexpression of ZNF146 has been established. Subcellular localization of ZNF146 protein in liver cells was studied by generation and expression of a green fluorescent protein (GFP) fusion protein. The nuclear localization and the reported DNA binding ability of ZNF146 protein provided a hint that ZNF146 may carry out its function in the cell system by interacting with specific genomic DNA sequences. Recombinant ZNF146 protein was expressed using bacterial and yeast system for the genomic DNA pull down assay in the identification of potential interacting genomic DNA sequences. Several potential genomic DNA sequences that interact with ZNF146 were identified and the gene MDM2 is the one of the candidates that is directly related to human carcinogenesis. MDM2 is a negative regulator of the tumor suppresser protein p53. Deregulation of MDM2 will impair the cell's ability in cell cycle arrest, DNA repair and apoptosis upon induced DNA damage. / Hepatocellular carcinoma (HCC) is a type of primary malignant liver tumor. And is one of the most frequent malignancies worldwide. The focus of this research project is the characterization of a Kruppel zinc finger protein, zinc Finger Protein 146 (ZNF146) using HCC as a disease model. The aim of this project is to understand the functional role ZNF146 and try to explore the mechanism of how ZNF146 might be involved in the carcinogenesis of HCC. / In order to have a better understanding with the protein ZNF146, SUMOylation properties of this protein has been studied. SUMO1 modification on ZNF146 has already been reported. And in our study, experimental result demonstrated that ZNF146 is also modified by SUMO2 and SUMO3 in liver cells. Other than the SUMOylation sites for SUMO1 protein which has been reported, modification sites for SUMO2 at the K247 and K275 positions were mapped, while K191R, K219R, K247R, K256R and K275R, five positions were mapped for SUMO3 modification. A more complete picture of the SUMOylation properties of ZNF146 has been revealed. Since we hypothesized that ZNF146 is related to the p53 tumor suppressor, cell cycle control and DNA repair pathway, a cell cycle study using flow cytometry was performed for the investigation of the effect on cell cycle regulation by ZNF146 overexpression. In our study, ZNF146 overexpression promoted the G1/S transition in the cell division cycle, which indicated that liver cells were more active for the progression of cell cycle. / On the other hand, using cDNA microarray technology expression profiles of ZNF146 overexpressing and non-overexpressing liver cell lines were compared and with real-time polymerase chain reaction, six candidate genes CRLF1, IFI44, ST6GAL1, LOC441601, IL18 and RAD17 were confirmed with their deregulation induced by the overexpression of ZNF146. Four of the candidates, IFI44, LOC441601, IL18 and RAD17 were found to be related to the p53 tumor suppressor activity or DNA damage, repair response and control. This observation, together with the result of genomic DNA pull down assay, gives us a hint that ZNF146 is possibly involved in liver carcinogenesis by affecting DNA repair and cell cycle control upon induced DNA damage. / The gene ZNF146 codes for a member of the Kruppel zinc finger proteins, however ZNF146 protein is different from most members of the Kruppel zinc finger proteins subfamily. It encodes a 33 kDa protein solely composed of 10 zinc finger motifs and is devoid of any non-zinc finger regulatory domain for interactions with other proteins. ZNF146 overexpression has been reported in a number of cancers including colon cancer and pancreatic carcinoma. However, the functional role of ZNF146 overexpression in tumorigenesis is yet to be solved and not much research on how ZNF146 might be invovled in the establishment of HCC was published. / To conclude, the experimental results of this study support the hypothesis that ZNF146 overexpression may deregulating the cell division cycle and some genes differentially regulated upon over-expression of ZNF146 are related to the regulations of DNA damage response. Future research on ZNF146 can be focused on the detail regulatory pathway of ZNF146 overexpression and its interaction between the p53 tumor suppressor, DNA damage response and cell cycle regulation, and a fuller picture of how ZNF146 overexpression might induce hepatocarcinogenesis can be revealed. / Yeung, Tsz Lun. / Adviser: Miu Yee (Mary) Waye. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3329. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 287-304). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A soja constitui uma das culturas mais importantes mundialmente, tanto social quanto economicamente. Consequentemente, informações moleculares sobre processos de desenvolvimento, bem como conhecimento detalhado das interações entre condições estressoras e a resposta da planta a fatores ambientais são necessários. A identificação e caracterização de genes que respondem a condições ambientais específicas constituem um passo inicial no entendimento dos processos adaptativos. Proteínas ricas em glicina (GRPs) são polipeptídeos contendo um grande número do aminoácido glicina em sua estrutura primária. Os genes codificantes de GRPs são regulados ao longo do desenvolvimento e regulados por auxina, ABA, frio, ferimentos, luz, ritmo circadiano, salinidade, seca, patógenos e encharcamento. Entretanto, há pouca informação sobre GRPs de plantas e seus papéis no desenvolvimento e resposta a estresses. As GRPs podem ser divididas em quatro classes (I, II, III, IV) de acordo com sua estrutura primária e presença de domínios característicos. A classe IV é composta por proteínas ligantes de RNA. Domínios adicionais permitem dividir a classe IV de GRPs em quatro subclasses (IVa, IVb, IVc, IVd). A subclasse IVc é representada por proteínas contendo um cold-schock domain (CSD) e dedos de zinco CCHC tipo retrovirais. O objetivo do presente estudo foi: (i) identificar e caracterizar os genes codificantes de classe IV de GRPs, (ii) verificar a padrão de expressão dos genes codificantes da subclasse IVc de GRPs e (iii) produzir plantas de soja transgênicas expressando o gene AtGRP2, o qual foi mostrado estar envolvido na floração e desenvolvimento da semente em Arabidopsis, e também poderia desempenhar um papel na aclimatação ao frio. Um total de 47 genes codificantes da classe IV de GRPs foi identificado no genoma da soja: 19 da subclasse IVa, sete da IVb, seis da IVc e 15 da IVd. Análises in silico indicaram uma expressão preferencial de todos os genes codificantes da subclasse IVc em tecidos em desenvolvimento. Análises de RT-qPCR revelaram que plantas jovens e maduras exibem uma expressão mais alta em folhas do que em outros órgãos, com exceção dos genes GRP2L_4/5 que tiveram expressão mais alta em sementes. GRP2L_4/5 e GRP2L_2 foram induzidos em resposta a baixas temperaturas. Sob estresse com ABA a expressão de todos os genes foi reprimida em folhas e/ou raízes, com exceção do gene GRP2L_2 que foi induzido em raízes. Em resposta a infecção com Phakopsora pachyrhizi, a expressão de GRP2L_2 e GRP2L_3 foi mais alta e precoce no genótipo suscetível quando comparada com o resistente, enquanto que a resposta de GRP2L_4/5 e GRP2L_6 foi mais tardia no genótipo resistente. Ainda, embriões somáticos secundários das cultivares Bragg, IAS-5 e BRSMG 68 Vencedora de soja foram usados para introduzir o gene AtGRP2 no genoma da soja por bombardeamento e sistema bombardeamento/Agrobacterium. Seis eventos de transformação independentes foram confirmados por PCR. No presente momento as plantas estão em desenvolvimento em frascos de vidro. No presente estudo a classe IV de GRPs em soja foi identificada e caracterizada. Este é o primeiro passo para elucidar o papel destas proteínas em plantas. / Molecular information on plant developmental process, as well as detailed knowledge of the interaction between stress conditions and plant response to environmental factors are essential for understanding the adaptive response. Glycine-Rich Proteins (GRP) have the amino acid glycine well represented in their primary structure. The genes encoding GRPs are developmentally regulated and induced by auxin, ABA, cold, wound, light, circadian rhythm, salinity, drought, pathogens, and flooding. However, there is scarce information about plant GRPs and its role on development and stress response. The GRPs can be divided into four classes (I, II, II and IV) according to their primary structure and the presence of characteristic domains. Class IV is composed by RNA-binding proteins. Additional domains permit to split class IV GRPs into four subclasses (IVa, IVb, IVc and IVd). Subclass IVc is represented by proteins containing a Cold-Shock Domain (CSD) and retroviral-like CCHC zinc fingers. The goal of the present study was: (i) to identify and characterize the genes encoding class IV GRPs, (ii) to verify the relative expression of genes encoding subclass IVc GRPs and (iii) to produce transgenic soybean plants expressing the AtGRP2 gene, which was shown to be involved in Arabidopsis flower and seed development, and can also play a role in cold acclimation. A total of 47 genes encoding class IV GRPs were found in the soybean genome: 19 from IVa, seven from IVb, six from IVc and 15 from IVd subclasses. In silico analyses indicated a preferential expression of all genes encoding subclass IVc GRPs in tissues under development. RT-qPCR analyses revealed that both young and mature plants exhibit relative higher expression of subclass IVc GRPs in leaves than in other organs, with exception of GRP2L_4/5 genes that have higher expression in seeds. The GRP2L_4/5 and GRP2L_2 were up-regulated in response to low temperatures. Under ABA stress the expression of all genes was down-regulated in leaves and roots, with exception of GRP2L_2 gene that was up-regulated in roots. In response to Phakopsora pachyrhizi infection, GRP2L_2 and GRP2L_3 expression was higher and earlier in the susceptible genotype when compared with that of the resistant one, while GRP2L_4/5 and GRP2_6 respond later in the resistant genotype. Furthermore, secondary somatic embryos of Bragg, IAS-5 and BRSMG 68 Vencedora soybean cultivars were used to introduce the AtGRP2 gene into the soybean genome by particle bombardment and bombardment/Agrobacterium system. Six independent Bragg transformation events were confirmed by PCR. In the present moment the plants are under development in glass flasks. In the present study the soybean class IV GRPs were identified and characterized. This is the first step to elucidate the role of these proteins in plants.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death. We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis. In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.

Oocyte development in Xenopus laevis spans six morphologically distinct stages (stage I-VI), and is associated with a decrease in protein O-GlcNAc levels. As a first step in elucidating the role of O-GlcNAc in developing oocytes, initial efforts were focused on isolation and identification of fifteen modified proteins that decrease during oocyte development. Stage I oocytes due to their high amounts of these proteins, were used as starting material for purification. Multiple affinity and specific antibody based purification technique were initially used in an attempt to enrich the O-GlcNAc proteins. Due to the unique properties of the proteins ultimately identified, these techniques were unable to provide sufficient material for sequencing. However, differential centrifugation coupled with 2D-gel electrophoresis was highly successful. The majority of isolated proteins were strongly basic in nature with pIs 8-10. Coomassie stained bands from 2D-analysis were trypsin digested, and peptides were sequenced by mass spectroscopy (Finnigan LCQ). Mass data were interpreted by Bioworks software, and protein sequences were compared to multiple protein databases. Initially, six proteins were identified as Thesaurin a (42Sp50), cytoplasmic mRNA binding protein p54, y-box homolog, Xp 54 (ATP dependent RNA helicase p54), Vg1 RNA binding protein variant A, Zygote arrest 1(Zar1) and Poly (A) binding protein (PABP). Thesaurin a, the main component of 42S particle of previtellogenic oocytes (stages I-III) is involved in tRNA storage and possess low tRNA transfer activity; y-box factor homolog and Xp54 are present in oocyte mRNA storage ribonucleoprotein particles; Vg1 RBP variant A associates mVg1 RNA to microtubules in order to translocate to the vegetal cortex; Zar1 is involved in oocyte-to-embryo transition; and PABP initiates mRNA translation. This study is the first to characterize these oocyte specific proteins as O-GlcNAc modified proteins. Overall, the presence of several O-GlcNAc proteins in oocytes, the reduction in their levels/ O-GlcNAc levels, and the variation in maturation time in the presence of HBP-flux modulators in developing oocyte indicates O-GlcNAc may play important roles in metabolism, cell growth and cell division of X. laevis oocytes. Therefore, identifying the remainder of these proteins and elucidating the O-GlcNAc role in their function is a worthwhile pursuit.

Non-protein coding eukaryotic genome sequences often referred to as junk DNA are estimated to encode several non-coding RNAs (ncRNAs) which may account for nearly 98% of all genomic output in humans. The output of such a wide spread transcription in eukaryotes consists of intronic, antisense and small RNAs. In addition to the classical ncRNAs such as rRNA, tRNA and small nucleolar RNAs, the eukaryotic genome encodes two distinct categories of ncRNAs, referred to as small ncRNAs and long mRNA–like ncRNAs (mlncRNAs). The long ncRNAs, which are transcribed by RNA Polymerase II, spliced and polyadenylated, are implicated in a number of regulatory processes such as imprinting, X-chromosome inactivation, DNA demethylation, transcription, RNA interference, chromatin structure dynamics and antisense mediated regulation. Expression of noncoding RNAs is altered during stress conditions and a large number of such transcripts have been identified of late. This study has identified a novel ncRNA whose expression is upregulated during viral infection of mouse brain. While we have named this RNA as VINC or virus inducible ncRNA, others have named it as NEAT1 (Hutchinson et al., 2007) and Men (Sunwoo et al., 2008). VINC/NEAT1/Men is associated with a distinct nuclear domain called paraspeckles Using a cell line as well as an animal model system we have investigated VINC in great detail and based on these studies we report that VINC is a nuclear ncRNA that localizes to paraspeckles and it interacts with the paraspeckle protein, P54nrb in both cell line model system as well as in animal tissues by a combination of in vitro and in vivo methods. We have also mapped the domains within VINC that are involved in P54nrb interactions. Till date, the only other RNA known to localise to paraspeckles is CTN-RNA. While CTN-RNA is a protein coding RNA, VINC does not code for a protein and thus VINC is the first ncRNA to be localized to paraspeckles. Further, the mechanism of nuclear retention of these two paraspeckle RNAs appears to be distinct. In case of CTN-RNA, it has been clearly shown that it is A-I edited and such hyperedited RNAs are retained by the p54/nrb mediated complex in nucleus (Zhang and Carmichael, 2001). However the mechanism by which VINC is retained in nucleus is not clear. There is apparently no A-I editing in VINC and hence VINC retention in the nucleus by binding to nuclear proteins such as p54/nrb might involve a different mechanism. It is well established of late that nuclear matrix retains RNAs and that there is a population of poly (A) RNA that is retained in nucleus (Huang et al.,1994 ; Carter et al.,1991). However the significance of such retention is not clear but it is believed that it might be important for some constitutive functions in nucleus (Nickerson et al., 1989). More investigations are needed to understand the exact functions of nuclear RNAs such as VINC in supporting the nuclear architecture. P54nrb is a multi functional nuclear protein that mediates most of its functions in association with PSF (Shav-Tal and Zipori, 2002). Phosphorylation status of P54nrb is a key determinant for its localisation to various nuclear regions. P54nrb is a multiphosphorylated protein during mitosis and its phosphorylation is mediated by PIN-1 at its C-terminus (Proteau et al., 2005). Tyrosine phosphorylation of P54nrb is essential for it to be retained in nuclear matrix (Otto et al., 2001). The N-terminal phosphorylation is speculated but not much has been investigated. The protein has two distinct RNA recognition motifs (RRMs) in its N-terminus that are responsible for its RNA binding activity. The significance of the p54/nrb-PSF heterodimer cannot be undermined as they have been shown to be important during HIV replication. The dimer is recruited by viral machinery and P54nrb has been shown to be exported to cytosol for binding to replicative complexes (Zolotukhin et al., 2003). During adenoviral replication in nucleus many SR proteins are recruited to viral replication foci and rearrangement of speckle components happen. It has been shown with respect to speckles that nuclear domains are highly dynamic and exchange of proteins depends upon the transcriptional status of cell (Lamond and Spector, 2003). Flaviviral replication complexes are hosted in nucleus and ~20% of this complex docks in nucleus and serves as an alternate site for viral replication. The presence of viral replicative complexes alters the nuclear organisation and hence modulation of gene expression is expected (Uchil et al., 2006). The up regulation of nuclear ncRNA such as VINC is definitively one of those events associated with viral replication and definitively one needs to study the various changes carefully to understand the role of VINC in virus life cycle and/or viral pathogenesis. VINC interaction with the multi-functional nuclear protein P54nrb raises interesting aspects related to function of P54nrb in JEV infection. Knockdown of P54nrb in human myeloid cell line results in abnormal size of paraspeckles and impairs chondrogenesis (Hata et al., 2008). PSF-P54nrb complex can divert many of HIV gag RNA complexes to paraspeckles thus trying to restrict viral replication. However the exact relationship between paraspeckles and its constituent proteins is not clear. The presence of ncRNA adds another new dimension to paraspeckles. It is unclear whether the ncRNA VINC is essential for paraspeckle structure but a recent study indicates that Men (VINC/NEATI) RNA may be essential for paraspeckle formation (Sunwoo et al., 2008). The exact function VINC in neuronal as well as non-neuronal cell nuclei remains elusive and more investigations are need to understand these aspects.

PTB (HnRNP I) is a multifunctional RNA binding protein which participates in a variety of RNA metabolic processes put together called as post transcriptional gene regulation. It interacts with shuttling hnRNPs L, K and E2 of the spliceosomal machinery and also with other RNA binding proteins like PSF, Raver1 and Raver2, which assists PTB in splicing. Based on the complexity of these processes and multifunctional nature of PTB, we hypothesized that; it might interact with various additional proteins not identified till date. Keeping this objective in mind, we set out to screen the custom made 18 day old mouse testes cDNA library in pGAD10 vector available in the laboratory, to hunt for novel interacting partners of PTB using the Clontech’s Matchmaker Gal4 yeast two hybrid system III. PTB1, the prototype of PTB was chosen and the above mentioned cDNA library was screened for novel PTB interacting partners. Twenty five large scale library transformations (spanning 8*106 independent clones) were performed and 99 putatives were obtained. By re-transformation of these library plasmids with bait construct to check for the interaction phenotype and eliminating bait independent activation of reporter genes and elimination of known false positives, only 5 clones were consistent with the interaction phenotype. All these library plasmids were sequenced with vector specific primers, ORF was identified and BLAST analysis for the identification of insert was done. Two of these clones encoded the partial CDS of mouse Protein Inhibitor of Activated STAT3-PIAS3. One of these encoded the partial CDS of mouse TOLL Interacting Protein-TOLLIP. The other two encoded the partial CDS of mouse importin-α and mouse hnRNP K, both of which were already known interacting partners of PTB. GST pull down assay and mammalian matchmaker co-immunoprecipitation was used for confirming the in vitro one to one physical interaction between PTB and these newly identified protein partners. Indirect Immunofloresence was used for demonstrating the co-localization of PTB and PIAS3 in Gc1Spg mouse spermatogonial cell line. The fact that PIAS3 an E3 SUMO ligase was picked up as an interacting partner of PTB was interesting and we hypothesized that PTB might be a sumoylation substrate. Towards this, we first resorted to the prediction of sumoylation consensus motif by using SUMOPLOT. PTB indeed was found to have sumoylation consensus sites. Subsequently, in vivo sumoylation of PTB was demonstrated, where in over expression of donor protein [SUMO-1] and acceptor protein [PTB] in RAG-1 mouse kidney cell line had resulted in the identification of an approximately 67 kDa slow moving SUMO modified myc tagged PTB band apart from the bulk of unmodified 57 kDa myc-PTB. This confirmed the fact that PTB is SUMO modified only at a single consensus target site in vivo and attempts are made to map this site of modification. SUMOylation regulates diverse biological processes in vivo ranging from nucleo- cytoplasmic shuttling, alteration of protein-protein interaction, DNA protein interaction etc. PTB shuttles rapidly between the nucleus and cytoplasm in a transcription sensitive manner and the translocation of PTB to the cytoplasm, happens under the conditions of cell stress, viral infections, apoptosis and exposure of cells to genotoxic agents like doxorubicin. Phosphorylation of PTB at Ser-16 residue has been shown to modulate the nucleo-cytoplasmic shuttling of PTB, albeit shuttling can also occur irrespective of this modification. Interaction of PTB with an E3 SUMO ligase-PIAS3 and the fact that it is SUMOylated in vivo, we hypothesize that K-47 residue present in the NLS/NES might be the most probable site of this SUMO modification and SUMOylation of PTB by PIAS3 might regulate the nucleo-cytoplasmic shuttling of PTB.

This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways. / QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA. / Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes. / Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.

The innate immune pathways that stimulate the expression of cytokines and proapoptotic factors in response to infection are triggered by the activation of the cytosolic receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5). Activation of both receptors occurs as a result of binding to RNA. MDA5 only recognizes double stranded forms of RNA, whereas RIG-I is capable of recognizing both single and double stranded RNA. In vivo, MDA5 is known to be stimulated by long (>1 kb) strands of RNA, forming filaments along the phosphate backbone. However, the manner in which MDA5 can recognize the terminal end of its RNA ligand is uncertain. I have examined the mechanism of binding of the MDA5 protein by comparing MDA5 binding to short (<18 bp) blunt RNA, 5’ triphosphate RNA, and RNA with a 3’ or 5’ overhang. It is shown that while the MDA5 protein regulatory domain (RD) is essential for RNA recognition, the MDA5 RD only weakly recognizes short double stranded RNA ligands with overhangs or a 5’ triphosphate group. The Cys951 residue was shown to disrupt stability of the MDA5 RD-RNA complex. Binding analyses were performed using a combination of SDS-PAGE, gel filtration analysis, and nondenaturing gel electrophoresis. In addition, structural data was gathered by crystallization of the MDA5 RD-RNA complex using X-ray crystallography. These results help to establish the manner in which MDA5 is regulated predominantly to the binding of long RNA ligands. Also included in this document is structural data on the dimer form of the PepQ protein from E. coli. PepQ is a highly conserved proline peptidase that has a secondary activity of hydrolyzing organophosphorus triesters, toxic compounds found in many pesticides. The PepQ protein was crystallized and analyzed by X-ray diffraction. The dimer interface was clearly defined within the structure and provides insight into how the active dimer forms from the PepQ monomer.