Current Research

Section III. What governs the changes in the parasite during differentiation: which genes are regulated and how are they controlled?

Trypanosomes are very unusual in the organization and regulation of genes in their genome. Instead of each gene being provided with its own promoter, many genes are co-transcribed within polycistronic (‘multi-gene’) transcription units. The genes within these clusters are usually not co-regulated, meaning that the abundance of their transcripts is unlikely to be controlled by the activity of the upstream promoter. Instead, genes must be regulated by ‘post-transcriptional’ mechanisms such as mRNA processing, mRNA stability, translation into protein and protein turnover. This extreme reliance upon the post-transcriptional control of gene expression is one of the interesting and very unusual biological processes in these parasites. One consequence is that trypanosomes have a very large numbers of RNA binding proteins.

We are particularly interested in small RNA binding proteins which are important in differentiation and stage-regulated gene expression. The first member of this family (TbZFP1) was identified as a molecule transiently enriched during synchronous differentiation between stumpy forms and procyclic forms. The protein is only 101 amino acids long, but contains an RNA binding motif (a CCCH zinc finger) and a protein interaction motif (PPPPY). Two other members of this family were subsequently identified by  genome analysis — TbZFP2 and TbZFP3. These are 139 and 130 amino acids long, respectively, contain a CCCH zinc finger and a WW protein interaction domain. This is of particular note because WW domains are known to interact with PPPPY domains. More recently, we have been characterising RNA binding proteins that drive the slender to stumpy transition (inncluding another small RNA binding protein, RBP7) or that repress stumpy gene expression in slender forms.

Using a combination of  gene knockout, RNA interference and ectopic over-expression we have dissected the role of each of these novel proteins in trypanosomes, this revealing how the genes are involved in stage-regulated processes.

TbZFP1 is responsible for the efficient morphological restructuring of the trypanosome cell during differentiation between bloodstream and procyclic forms. Particularly, genetic knockout of TbZFP1 eliminates the repositioning of the mitochondrial genome characteristic of this life-cycle transition.

TbZFP2 contributes to the initiation of differentiation, with RNA interference for this molecule  preventing transition between bloodstream and procyclic forms. Interestingly, over-expression of TbZFP2 causes an extreme extension at the posterior end of the trypanosome cell by microtubule outgrowth. We interpret this phenotype (which we call ‘nozzle’ formation) as an over-exaggeration of the normal cytoskeletal outgrowth that occurs during differentiation.

TbZFP3 is similar to TbZFP3 in its overall molecular architecture. Moreover, when over expressed, this molecule enhances differentiation between life cycle stages and causes nozzle formation in procyclic forms.

TbRBP7 is an RRM class RNA binding protien that promotes stumpy formation when overexpressed and which reduces stumpy formation when ablated by RNAi.

Therefore, each protein regulates developmental events, and we are investigating the transcripts that they regulate and the moelcular machinery through which they operate.

For more information on this theme, see:

Hendriks, E. and Matthews, K. R. (2005)
Disruption of the developmental programme of Trypanosoma brucei by genetic ablation of TbZFP1, a differentiation-enriched CCCH protein. Molecular Microbiology  57: 706-716.
Hendriks, E.; Robinson, D.R. , Hinkins, M and Matthews, K.R. (2001).
A novel CCCH protein which modulates differentiation of Trypanosoma brucei to its procyclic form. EMBO J. 20:6700-6711.
Paterou A, Walrad P, Craddy P, Fenn K, Matthews K. (2006)
Identification and stage-specific association with the translational apparatus of TbZFP3, a ccch protein that promotes trypanosome life cycle development. J Biol Chem. 281(51):39002-13
Walrad P, Paterou A, Acosta-Serrano A, Matthews KR (2009).
Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.
PLoS Pathog. 2009 Feb;5(2):e1000317.

Walrad, Pegine; Capewell, Paul; Fenn, Katelyn and Keith R. Matthews (2011)

The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei.

Nucleic Acids Research PMID: 22140102

Binny M. Mony*; Paula MacGregor*; Alasdair Ivens, Federico Rojas, Andrew Cowton, Julie Young, David Horn and Keith R. Matthews (2013)

Genome wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei

Nature doi:10.1038/nature12864

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