[PDF] Functional Characterization Of Two Rna Binding Proteins P34 And P37 From Trypanosoma Brucei eBook

Author : Kimberly M. Prohaska
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Page : 211 pages
File Size : 23,35 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 : 44,89 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.

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Page : 202 pages
File Size : 27,96 MB
Release : 2006
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Two homologous proteins, p34 and p37, are developmentally regulated in Trypanosoma brucei, with p34 primarily expressed in procyclic form and p37 exclusively expressed in bloodstream form. Previous studies have shown that the mRNA stability of p34 is decreased in bloodstream form, while the translation efficiency and protein stability of p37 are decreased in procyclic form. The transcripts of p34 and p37 contain divergent sequences in their 3' untranslated regions. Using reporter gene analysis, we have shown that the p34 3' UTR decreased reporter mRNA stability in bloodstream form. Deletion of a 17-nt AU rich element (ARE) from the p34 3' UTR enhanced the mRNA stability. The ARE itself, when inserted into a control 3' UTR sequence, did not regulate gene expression. Secondary structure analysis and RNase sensitivity assays showed that the 17-nt ARE is located in a single-stranded region in the p34 3' UTR. Deletion of this ARE decreased the size of the single-stranded region and the sensitivity to RNase A. The reporter gene analysis also showed that the p37 3' UTR developmentally regulates translation efficiency. Deletion of a 29-nt polypyrimidine sequence from the p37 3' UTR did not change the expression of reporter luciferase. However, insertion of the 29-nt sequence in control 3' UTR decreased the expression of reporter luciferase in both life stages of T. brucei. Secondary structure analysis indicated that deletion of the 29-nt sequence did not change the structure of the p37 3' UTR, which forms a long stem structure with nearly all of the 3' UTR sequences residing in a double stranded region. The insertion of the 29-nt sequence alone moderately modified the structure of the control 3' UTR, causing a short U rich sequence to reside in a single stranded region. All these results indicate that the secondary structures are involved in gene regulation controlled by the 3' UTRs of p34 and p37. We have identified proteins that bind to the p34 and/or p37 3' UTR. T. brucei HMG like TDP-1 protein binds to both 3' UTRs without poly (A) tails, while addition of a poly (A) tail inhibited binding of TDP-1 to the p37 3' UTR. TDP-1 binds to the mRNAs of p34 and the T. brucei phosphoglycerate kinase (PGKB) gene but not to the p37 mRNA. Both PGKB and p34 are regulated at the mRNA stability level by AREs in the 3' UTRs. These results indicated that the TDP-1 protein is involved in regulation of mRNA stability. In vitro RNA degradation assays have shown that TDP-1 protected RNA with the p34 3' UTR sequences from degradation in T. brucei bloodstream cell extracts. A second protein, glycerol kinase (GLK) from T. brucei was shown to bind RNAs with U rich sequences. The GLK protein showed higher affinity to the p37 3' UTR compared to the p34 3' UTR.

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Page : 156 pages
File Size : 26,73 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.

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Page : 88 pages
File Size : 38,23 MB
Release : 2007
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Pentatricopeptide repeat proteins (PPRs) are one of the largest newly identified protein families. PPR is characterized by the presence of a tandemly repeated 35 amino acids motif. It is extremely abundant in plants, and found in many organisms including mammals. PPRs are said to play essential roles in mitochondria, probably via binding to organelle transcripts. A number of recent reports characterizing PPR proteins indicate that PPR are important regulatory proteins that play essential roles in polycistronic RNA processing, RNA stability, and translation of special transcripts [2, 64]. To date, there are 23 PPRs identified in T.brucei by Mingler et al. [2] and 28 distinct PPRs detected by Pusnik et al. [1] by using different bioinformatics approaches. T.brucei encodes far more PPRs than the other non-plant organisms such as mammals. Trypanosomes undergoes a complex life cycle and thus it need to adapt to the changes of environment where it resides. T.brucei undergoes different morphological forms, metabolic changes, and alterations in gene expression during the changes of environment between insect and mammalian hosts. Thus, T.brucei serves as a good model to study the function of PPRs. We utilized tetracycline (tet)-regulated RNA interference (RNAi) to study the function of two T.brucei PPR proteins, TbPPR6 and TbPPR7. Northern blot analysis shows that TbPPR6 (Tb11.01.7930) was down-regulated by 49% while TbPPR7 (Tb927.3.4550) showed a decrease level of 23% in tetracycline induction RNAi cells. Knock-down of TbPPR6 caused a slow growth phenotype, while TbPPR7 knock-down caused only a minor slow growth phenotype, suggesting that TbPPR6 and TbPPR7 are essential for optimal growth of T.brucei . Poisoned primer extension assays demonstrated that depletion of TbPPR6 and TbPPR7 to the levels described above did not affect the mRNAs analyzed. However, there may be a modest effect on COI stability, particularly after depletion of TbPPR7. To characterize the RNA binding properties of TbPPR4 (Tb10.70.5780), recombinant TbPPR4 full length (PTbPR4-FL), and two of its truncated versions (TbPPR4-1, TbPPR4-0) were expressed and purified. Homopolymer binding assays show that TbPPR4-FL preferentially binds poly-(G) agarose beads over the other homopolymers. We further characterized the TbPPR4-RNA interaction by changing the salt concentration (NaCl) of the binding buffer. Increasing salt concentration in the binding buffer decreases the binding ability of TbPPR4-FL to poly-(G) agarose beads, indicating that electrostatic interactions play a prominent role in the binding of TbPPR4-FL. Interactions such as hydrogen bonding and hydrophobic interactions may also involve in the interactions of TbPPR4-FL with poly-(G). Titration experiments of TbPPR4-FL, TbPPR4-1, and TbPPR4-0 show that TbPPR4-FL binds significantly better than TbPPR4-1 and TbPPR4-0 while TbPPR4-1 binds better than TbPR4-0 to poly-(G). Thus, PPRs with increasing numbers of PPR motifs have higher affinity to RNA. Our studies add to the growing literature that defines PPR motifs as RNA binding motifs, and suggest that TbPPR6 and TbPPR7 maybe play a role in mitochondrial RNA processing and/or stability.

Author : Martin Ciganda
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Page : 221 pages
File Size : 34,95 MB
Release : 2012
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ItalicTrypanosoma brucei/italic is the causative agent of human African trypanosomiasis. It belongs to the kinetoplastids, a group of early-branching eukaryotes with many unique biochemical and genetic characteristics. Our laboratory had previously identified two abundant RNA binding proteins specific to the parasitic trypanosomatids T. brucei, T. cruzi and Leishmania. These proteins, P34 and P37, are essential in T. brucei. Previous data indicated that P34 and P37 associate with 5S rRNA in the nucleus. In silico analysis of the T. brucei 5S rRNA yielded two different structures with similar energetic parameters. Both structures are organized in five stems and five loops. One of them has a typically eukaryotic Loop C, while the other has an unusually reduced Loop C. RNase H digestion patterns were consistent with a form in which loop C is exposed and available for inter-molecular interactions.^Studies using recombinant proteins demonstrated that both P34 and P37 can bind 5S rRNA with high specifity and affinity in the absence of additional cellular factors. Different approaches were taken to investigate which domains on the 5S rRNA are involved in the interaction with P34 and P37. We conclude that the Loop A / Stem V region in 5S rRNA is an important interaction site for the association with P34 and P37. However, upon binding, both P34 and P37 protect a large area of the molecule which extends to Loop C. In other eukaryotes, the Loop A / Stem V region is involved in an interaction with TFIIIA, a factor that has not been identified in the T. brucei databases. Loop C mediates a high affinity, conserved interaction with ribosomal protein L5. This association serves to stabilize the nascent 5S rRNA, and transport it to the nucleolus. The L5-5S rRNA RNP is the only characterized pre-ribosomal particle in eukaryotes to date. In silico studies of the T.^brucei L5 protein reveal deviation from the eukaryotic consensus at potentially key positions implicated in binding to 5S rRNA. In vivo studies demonstrate that the T. brucei L5 protein binds a lower percentage of total 5S rRNA than previously characterized L5 proteins. In addition, in vitro studies with recombinant proteins show that the affinity of T. brucei L5 for 5S rRNA is 20-fold lower than that of the Xenopus laevis L5 protein. A mutation in the T. brucei C-terminus domain of L5 that restores a positive charge within an alpha helix to consensus increases binding to 5S rRNA. In T. brucei cells lacking P34 and P37, 5S rRNA is destabilized and ribosomal biogenesis is impaired. A similar phenotype is observed in yeast cells depleted of L5. We hypothesize that in trypanosomes, L5 is less efficient at binding 5S rRNA, and that P34 and P37 have been recruited to compensate L5 function.