The Effects of Polysomal mRNA Association and Cap Methylation on Gene Expression in Trypanosoma brucei

Abstract

Contrasting physiological requirements for T. brucei survival between procyclic (vector) and bloodstream (mammal) forms necessitate different molecular processes and therefore changes in protein expression. Transcriptional regulation is unusual in T. brucei because the arrangement of genes is polycistronic; however, genes which are transcribed together are subsequently cleaved into separate mRNAs by trans-splicing and are individually regulated. During the process of trans-splicing, a 39-nucleotide splice-leader RNA is added to the 5´ end of mRNA. In this study, gene regulation in trypanosomes will be examined in the context of the 7-methylguanosine cap attached to the 5´ end of the splice-leader. Interestingly, in addition to the capping enzymes identified in other eukaryotes, trypanosomatids have an additional guanylyltransferase and methyltransferase in the form of a bifunctional enzyme (TbCGM1). TbCGM1 was found to be essential in bloodstream form T. brucei, although the purpose of this bifunctional capping enzyme remains unclear. Null mutants of a related enzyme, monomeric methyltransferase TbCMT1, did not show an effect on cell viability in culture, however, the enzyme proved to be important for virulence in vivo. Complementary to the study of T. brucei capping enzymes, we worked to develop a method to allow structural analysis of the 5´mRNA cap by mass spectrometry.
Following pre-mRNA processing, regulation of the mature mRNAs is a tightly controlled cellular process. While multiple stage-specific transcripts have been identified, previous studies using RNA-seq found that the changes in overall transcript level do not necessarily reflect the abundance of the corresponding proteins. We hypothesized that in addition to mRNA stability, mRNA recruitment to ribosomes may play a significant role in the regulation of gene expression in T. brucei. To approach this question, we performed RNA-seq of total, subpolysomal, and polysomal mRNA. This transcriptomic data was then correlated with published proteomic studies to obtain a global picture of the relative translation efficiencies and their relationship to steady-state protein levels between bloodstream and procyclic form T. brucei.

Date of Award	2014
Original language	English
Awarding Institution	University of Dundee
Supervisor	Michael Ferguson (Supervisor) & Victoria Cowling (Supervisor)