Translational control and the escape from translational arrest in stumpy form Trypanosoma brucei
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Date
30/11/2012Author
Monk, Stephanie Lydia Spencer
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Abstract
The transmission of Trypanosoma brucei, the causative agent of human African
trypanosomiasis, depends upon the development in the bloodstream of 'stumpy
forms' from non-transmissible 'slender forms'. In stumpy forms many mRNAs are
downregulated and translation is generally repressed. However, a small subset of
genes escape this repression and are upregulated, presumably as an adaptation for
transmission. To understand the basic of this, regulatory sequences within the 3'UTR
of a major stumpy-enriched transcript (an ESAG9 gene) have been characterised.
This identified a signal responsible for gene silencing in slender forms and gene
activation when cells develop to stumpy forms.
An investigation was made of upstream open reading frames (uORFs) as a
mechanism for the control of stumpy form gene expression. No evidence was found
of uORF control, but one gene investigated was found to produce two transcripts
through trans-splicing at different sites. These transcripts, which were found to
exhibit some differential abundance between life-cycle stages, would generate a long
and short form (from an internal ATG) of the encoded protein. Both are predicted to
contain a UBA/TS-N (ubiquitin associated) domain, however, the longer form of the
protein is also predicted to contain a transmembrane helix and cleavable signal
peptide, suggesting a different localisation. However, ectopic expression of either
protein form with a Ty epitope tag resulted in the same protein localisation.
Additionally, the transcripts of two translational protein homologues, TbeIF4E4 and
TbeIF6, were identified as upregulated in stumpy forms. Radiolabelled-methionine
experiments and polysome analysis showed that overexpression or RNAi-mediated
ablation of TbeIF6 resulted in a decrease in protein synthesis and decrease in
translation. Unlike its archaeal homologue, TbeIF6 protein was not induced by coldshock
treatment.
Finally, to identify which transcripts escape translational repression in stumpy forms
an analysis was made of polysome-associated transcripts by RNA-sequencing. This
identified potentially interesting genes for further investigation, and showed that
many procyclic-enriched transcripts were also enriched in stumpy form polysomeassociated
RNA, confirming these cells as preadapted for transmission. Together,
this work has characterised a 3’UTR regulatory element in a stumpy-enriched
transcript, examined alternative trans-splicing of another transcript, investigated two
translational protein homologues and identified transcripts that escape translational
repression in the transmissible life-cycle stage of T. brucei.