[PDF] Identification And Characterization Of A Guide Rna Binding Protein From Trypanosoma Brucei Mitochondria eBook

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Page : 156 pages
File Size : 45,34 MB
Release : 2008
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The protozoan parasite and causative agent of human and animal African trypanosomiasis, Trypanosoma brucei, is a leading cause of morbidity and mortality among epidemic rural regions of sub-Saharan Africa. T. brucei is transmitted to the bloodstream of the mammalian host by the tsetse fly, where it resides extracellularly and evades immune detection by a mechanism called antigenic variation. Due to the antigenic nature of the parasite, the prospect for vaccine development is grim. Instead, disease treatment relies solely on chemotherapeutic strategies that target the unique and exploitable biology of the trypanosome. Such unique aspects of T. brucei biology include polycistronic transcription, pre-mRNA trans -splicing, and kinetoplastid RNA editing. The developmental regulation of trypanosome gene expression relies on coordinated post-transcriptional events in which RNA binding proteins play a leading role. With this in mind, our focus is directed towards a family of protein arginine methyltransferases (PRMTs), which are implicated in a variety of post-transcriptional events and hypothesized to play a regulatory role in trypanosome gene expression. Protein arginine methylation is a post-translational modification that modulates the function of a variety nucleic acid binding proteins, impacting transcriptional and post-transcriptional gene expression. These modifications are catalyzed by a family of PRMTs described in mammals and yeast for which five homologs have been identified in the T. brucei genome. The initial characterization of TbPRMT1 and TbPRMT5 has been explored, the latter of which is described in Chapters II and III of this thesis. TbPRMT1 is a type I PRMT and catalyzes asymmetric dimethylarginine modifications, while TbPRMT5 is a type II PRMT and catalyzes symmetric dimethylarginine modifications. Both TbPRMT1 and TbPRMT5 are constitutively expressed in bloodstream and procyclic form trypanosomes, localize to the cytoplasm, and are not essential for growth. Both enzymes methylate a variety of substrates in vitro, including the mitochondrial regulatory protein, RBP16. In addition, TbPRMT5 was shown to associate in vivo with a kinetoplastid-specific nucleotidyltransferase and an ATP-dependent RNA helicase, the latter of which is an in vitro TbPRMT5 substrate. In vivo analysis of higher-order TbPRMT5-TAP-containing complexes indicates that TbPRMT5 associates with two predominant protein complexes with molecular weights of approximately 250 and 700 kDa. The latter of these complexes is unstable and does not withstand glycerol gradient fractionation. While TbPRMT1 and TbPRMT5 do not play a global role in trans -splicing or decay of nuclear encoded RNAs, they both play a role in mitochondrial gene expression. In Chapter III of this dissertation, I show that disruption of TbPRMT5 in procyclic form trypanosomes by RNA interference results in the destabilization of never edited COI and ND4 and both edited and unedited apocytochrome b (CYb) and COII mitochondrial RNAs (Chapter III). In addition, TbPRMT5 disruption resulted in a modest but significant increase in the steady-abundance of mitochondrial-encoded guide RNAs (gRNAs), suggesting that TbPRMT5 plays a role in gRNA turnover. Furthermore, I demonstrate that TbPRMT5 potentially modulates the function of at least four mitochondrial methylproteins. Several in vivo hypomethylation defects were observed in mitochondrial lysates of TbPRMT5-disrupted cells, and TbPRMT5 appears to affect the expression and/or mitochondrial localization of one putative in vivo substrate. The characterization of TbPRMT5 substrates, as well as the remaining T. brucei PRMTs, is currently being explored. In summary, I provide evidence that TbPRMT5-catalyzed arginine methylation plays important regulatory roles in T. brucei mitochondrial gene expression, and its association with RNA modifying enzymes suggests that it likely impacts additional specific RNA metabolic events in the nucleus and/or cytoplasm.

Author : Kimberly M. Prohaska
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Page : 211 pages
File Size : 45,59 MB
Release : 2008
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Our laboratory has previously identified and purified two nearly identical RNA binding proteins, P34 and P37, from Trypanosoma brucei. The only differences between these two proteins are an 18 amino acid insert in the N-terminus of P37 and four single amino acid differences with respect to P34. Initial experiments were performed to elucidate molecular binding partners of P34 and P37, which demonstrated interactions with a family of nucleolar phosphoproteins, NOPP44/46, ribosomal protein L5, and 5S rRNA. To further characterize the association of P34 and P37 with NOPP44/46, a P34/P37 RNAi cell line was utilized, in which expression of both proteins was simultaneously knocked down. In the absence of these proteins, NOPP44/46 nuclear protein levels increased approximately 12-fold, suggesting a role for P34 and P37 in regulating NOPP44/46 expression. It was then demonstrated that P34 and P37 do not regulate NOPP44/46 at the level of transcript abundance or stability, or at the level of total cellular protein. Results from immune capture experiments showed that P34 and P37 associate with exportin 1, a nuclear export factor and, that they mediate an association between this protein and NOPP44/46, thereby regulating their cellular localization. Since NOPP44/46, L5, and 5S rRNA are involved at some stage during biogenesis of the 60S ribosomal subunit and each associates with P34 and P37, it was hypothesized that P34 and P37 are also involved in this pathway. Immune capture experiments specific for ribosomal proteins which enter the 60S biogenesis pathway at different points were performed to determine when and where P34 and P37 come into the pathway. The results from these experiments showed that P34 and P37 enter into the pathway at the early 90S pre-ribosomal particle in the nucleolus and that they remain associated subsequent to nuclear export and subunit joining. Interestingly, experiments performed using the P34/P37 RNAi cells demonstrated that in the absence of P34 and P37, the 60S subunit no longer interacts with XpoI or Nmd3, components of the nuclear complex required for the export of the yeast 60S subunit. These results support an essential role for P34 and P37 in the nuclear export of the 60S subunit in trypanosomes. Together, the results presented in this thesis demonstrate the potential for multi-functional roles for P34 and P37 in the ribosomal biogenesis pathway. These studies lay the groundwork for further experiments aimed at more specifically determining the function(s) of P34 and P37 in ribosomal biogenesis.

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Page : 164 pages
File Size : 44,37 MB
Release : 2006
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We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, from Trypanosoma brucei . These proteins have been shown to bind 5S rRNA and a family of nucleolar phosphoproteins, NOPP44/46. In the studies presented here, we have employed RNA interference studies in order to gain further insight into the protein-protein and protein-RNA interactions of p34 and p37 in T. brucei . Loss of p34 and p37 results in disruption of a higher molecular weight complex containing 5S rRNA, as well as a dramatic decrease in 5S rRNA levels suggesting that these proteins serve to stabilize 5S rRNA. No change in ribosome assembly was found although a significant decrease in overall protein synthesis occurred within p34/p37 RNAi cells. We next evaluated the relationship of p34 and p37 with other conserved 5S rRNA binding proteins. We showed that p34 and p37 do not stably interact with the La protein although this protein is able to form a higher molecular weight complex(es) in the absence of p34 and p37. La protein levels exhibited a modest increase in p34/p37 RNAi cells. Loss of p34 and p37 and subsequent loss of 5S rRNA does not effect the participation of the L5 ribosomal protein in complex formation or L5 protein levels. We found that p34 and p37 bind to the L5 ribosomal protein. The amount of 5S rRNA bound to p34 and p37 is similar to the amount bound by L5. The loss of p34 and p37 in our RNAi cell lines also led to disruption of a higher molecular weight complex containing the NOPP44/46 proteins as well as a dramatic 12-fold increase in NOPP protein levels within the nucleus. No changes occurred in either NOPP44/46 mRNA steady state levels or stability indicating that p34 and p37 do not affect NOPP expression post-transcriptionally. Surprisingly, we found no alterations in NOPP protein levels in total cell extracts from p34/p37 RNAi cells, in sharp contrast to the increase in NOPP nuclear extracts from these same cells. These results have led us to propose that p34 and p37 function in the regulation of NOPP44/46 intracellular localization.

Author : Carmen Priscila Pena Diaz
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Page : pages
File Size : 32,77 MB
Release : 2011
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The Mitochondrial Carrier Family encloses a group of transmembrane proteins that transport metabolites across the mitochondrial inner membrane. The ADP/ATP carrier is the most widely studied MCF protein. It catalyzes the counter exchange of ADP for ATP in the mitochondrion of all eukaryotes. In the genome of the kinetoplastid parasite Trypanosoma brucei, three putative ADP/ATP carrier sequences (MCP5, MCP15 and MCP16) and one GDP/GTP (MCP13) entries were analyzed by sequence analyses and phylogenetic reconstruction. AACs phylogenetic reconstruction proved a strong association with yeast, funghi and plant clades, whilst separates from those AACs and from metazoans. MCP13 groups with GGCs, seems to be present only on lower eukaryotes and do not seem to present any homologues in metazoans. Gene deletion studies were performed to assess the roles of MCP5, MCP15, MCP16 and 13. A conditional double knockout cell line, with an inducible myc-tagged rescue copy was constructed for MCP5, which proves the essentiality of the protein for the parasite. Growth curves of the mutant cell line proved a growth defect phenotype in various carbon sources conditions. Mitochondrial ATP production assays were performed in the mutant cell line, in presence and absence of the inducible protein, using permeabilized cells with digitonin that confirmed the ADP/ATP transport activity of the carrier. For invitro activity assays, the carriers were cloned and expressed in Escherichia coli and Spodoptera frugiperda, solubilised and reconstituted into liposomes. Unfortunately, the reconstitution was unsuccessful and the conditions and methodologies are discussed.

Author : H. Ulrich Göringer
Publisher : Springer Science & Business Media
Page : 239 pages
File Size : 29,5 MB
Release : 2008-01-12
Category : Science
ISBN : 3540737871

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Goringer’s brilliant new work dedicates a chapter to each of the main types of RNA editing – the very first volume to do so. All of the sections here have been written by experts in the various research areas and a specific focus is put on the correlation between RNA structure and function, as well as on the complex cellular machineries that catalyze the different editing reactions. This leads to a "state of the art" compendium of our current knowledge on RNA editing.