[PDF] Characterization Of The Rna Binding Protein Rbp10 In Trypanosoma Brucei eBook

Author : Kimberly M. Prohaska
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Page : 211 pages
File Size : 23,55 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 : 18,45 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 : 36,80 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.

Author : Zhiquan Lu
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Page : pages
File Size : 40,23 MB
Release : 2015
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"Trypanosomes are protozoan parasites that alternate between insect and mammalian hosts over the course of their life cycle. In the absence of transcriptional control, these parasites depend almost entirely on post-transcriptional mechanisms to remodel gene expression in response to internal and external stimuli. This is achieved through a complex network of trans-acting RNA-binding proteins (RBPs) and cis-acting regulatory elements. Our computational studies identified highly conserved adenosine-uridine (AU)-rich elements (AREs) in the 3' untranslated regions (UTRs) of a large number of Trypanosoma brucei mRNAs. These ARE-containing transcripts are up-regulated in the stationary phase of in vitro-cultured procyclic form cells and down-regulated in stumpy and slender bloodstream form cells, suggesting a role for AREs in the regulation of parasite differentiation. The expression of ARE-containing transcripts positively correlated with three RBPs identified using sequence analysis as potential remote homologs of ELAV (Embryonic Lethal Abnormal Vision) proteins. In higher eukaryotes, proteins of the ELAV-like family regulate gene expression by stabilizing or promoting degradation of ARE-containing transcripts, suggesting a similar role for these proteins in regulating trypanosomatid ARE-containing mRNAs. Using in vitro electrophoretic mobility shift assays (EMSA) and RNA immunoprecipitation followed by deep sequencing (RIP-seq), we confirmed that the T. brucei ELAV-like proteins associate specifically and directly with ARE-containing transcripts. Furthermore, microarray analysis of over-expression and RNA interference (RNAi) knockdown cell lines suggests that the three identified ELAV-like proteins (ELAV-LPs) specifically regulate expression of ARE-containing transcripts. Understanding the role of the potential ELAV-LPs in the complex network of post-transcriptional regulation in T. brucei may elucidate new pathways and potential therapeutic targets for the treatment of trypanosomiasis." --