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Exploring the biochemical roles of TbeIF4E-3 AND TbeIF4E-5 in T. brucei cell cycle

Rubus, Jacob P (2015) Exploring the biochemical roles of TbeIF4E-3 AND TbeIF4E-5 in T. brucei cell cycle. Master's Thesis, University of Pittsburgh. (Unpublished)

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African Sleeping Sickness afflicts millions of people in sub-Saharan areas of the continent. The disease causing agent, the unicellular eukaryotic protozoan Trypanosoma brucei, is intensely studied, yet no safe cure has been developed and has become a public health issue. Because gene expression control in T. brucei exhibits unique patterns, it is attractive for drug targeting. Our lab aims to explore the cellular and molecular role(s) of TbEIF4E-3 (4E-3p) and TbeIF4E-5 (4E-5p), structurally divergent homologs of eukaryotic translation initiation factor 4E (eIF4E), a canonical mRNA 5’ cap binding protein involved in post-translational regulation of gene expression. The 4E-3 and 4E-5 proteins are essential in cell cycle in trypanosomes. We hypothesize 4E-3p and 4E-5p to be key regulons, proteins that regulate the translation of distinct sets of multiple, functionally related mRNAs required for normal cell cycle. Evidence from immunofluorescence microscopy (IMF) by other labs and our own revealed cytoplasmic localization of 4E-3p in mRNP granules. Tandem co-immunoprecipitation (co-IP) and mass spectrometry (MS) revealed 4E-3p association with canonical protein partners found in other eukaryotes known to be involved in translation initiation and control such as eIF4G and eIF4A, consistent with earlier studies. Significantly, this present work further identified unique uncharacterized polypeptides, implying both conserved as well as novel functions. Additionally, RNAseq analysis from co-IPs revealed that 4E-3p also associates with various mRNAs, some of which are involved in the cell cycle, providing evidence towards potential translational regulation of cell cycle. By contrast, 4E-5p appeared more associated with the mitochondria from IMF studies, consistent with findings by others. Combined co-IP and MS analysis showed association with translational regulators, whereas RNAseq revealed association with mRNAs regulating cell division and replication control distinct from those associated with 4E-3p. These findings suggest that both 4E-3p and 4E-5p may act separately to regulate cell cycle, replication, and gene expression in T. brucei in a manner consistent with a proposed Parallel RNA Regulon Model. Among potential drug targets being analyzed are translational regulating proteins, of which 4E-3p and 4E-5p are attractive candidates due to their discovered role in normal cell cycle.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Rubus, Jacob Pjpr25@pitt.eduJPR25
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberNorris, Karen Akan1@pitt.eduKAN1
Committee MemberReinhart, Todd A.reinhar@pitt.eduREINHAR
Committee MemberMartinson, Jeremy Jjmartins@pitt.eduJMARTINS
Thesis AdvisorTarun,
Date: 29 June 2015
Date Type: Publication
Defense Date: 14 April 2015
Approval Date: 29 June 2015
Submission Date: 6 April 2015
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 138
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: TbeIF4E-3, TbeIF4E-5, trypanosome, trypanosoma brucei, T brucei,
Date Deposited: 29 Jun 2015 14:38
Last Modified: 15 Nov 2016 14:27


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