Regulation of Gene Expression in Parasitic Protozoa

The quality of life of millions is compromised by parasitic infection. Trypanosomes, spread by tsetse flies to humans and grazing animals in sub- Saharan Africa or by reduviid bugs in many areas of South and Central America, cause debilitating and lethal disease. These unicellular, eukaryotic organisms reside extracellularly, in the bloodstream and tissue spaces of the host or intracellularly, within smooth muscle cells. As parasites, they readily differentiate during their complex life cycle to adapt to numerous nutritional conditions and immunological challenges. Scientific research has uncovered a multitude of unusual genetic, biochemical and structural properties of trypanosomes. We hope that by exploiting some of these parasite-specific traits we can devise ways to alleviate disease.

Messenger RNA molecules in eukaryotic cells usually begin as large precursor forms that are cut and spliced to produce stable and translatable mature mRNAs. In most cases, a single molecule of RNA is the starting material. However, trypanosomes synthesize messenger RNAs in a unique way. All trypanosome mRNAs are produced by a splicing reaction that fuses a short, capped RNA (called the spliced leader) to each protein coding pre-mRNA. The spliced leader is essential for gene expression; however, its synthesis, regulation and role in mRNA maturation and translation remain enigmas. Our work is focused on understanding the nucleic acid and protein signals responsible for these processes.

To dissect the components of the spliced leader gene essential for its transcription, we are using a combination of genetic analysis and DNA-protein binding studies. Protein purification and subsequent genetic analysis will enable us to identify unique trans-acting factors of the spliced leader transcription pathway. This work has lead to our discovery of PBP-1, a basal transcription factor that is an essential component of the spliced leader RNA gene expression pathway.

Since the 5' cap present on each mature mRNA is added in trans in trypanosomes, the "one pre-mRNA -one mRNA" rule observed in other eukaryotes does not apply here. Thus, trypanosomes synthesize polycistronic pre-mRNAs that are cleaved into multiple mature mRNAs via RNA trans-splicing. Post-transcriptional regulation of gene expression is probably orchestrated in part by differential spliced leader addition to specific sites within polycistronic pre-mRNAs. We are investigating the signals that direct the spliced leader to specific precursor transcripts whose mRNA levels appear to be post-transcriptionally regulated.

Finally, we have developed an in vitro RNA turnover system that is enabling us to explore and define the signals that regulate differential mRNA turnover as a function of life cycle stage in trypanosomes.

Selected Publications

• Das, A. and Bellofatto, V. (2003) RNA polymerase II dependent transcriptions in trypanosomes is associated with a SNAP complex like transcription factor. Proc. Natl. Acad. Sci. USA. 100:80-85.
  
  
• Das, A. and Bellofatto, V. (2004) Genetic Regulation of Protein Synthesis in Trypanosomes. Current Molecular Medicine, 4, 577-584.
  
  
• Gilinger, G., Luo, H. and Bellofatto, V. (2004). Invivo transcription analysis of trypanosome transcription using chromatin immunoprecipation. Molecular & Biochemical Parasitology, 134,169-173.
  
  
• Milone, J., Wilusz, J. and Bellofatto, V., (2004) Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p RNA, 10, 448-457.
  
  
• Bergman, N., Milone J., Bates, E., Opyrchal, M., Bellofatto, V. and Wilusz, J. (2004). Assessing mRNA decapping in Cellular Extracts. In:? mRNA processing and metabolism: Methods and Protocols. Methods Mol Biol, 257,181-192.