Laboratory of RNA Biology of Protists
Head: RNDr. Zdeněk Paris, PhD.
Joined the BCAS: February 2014,
2011–2014: postdoctoral researcher at the Ohio State University, Columbus, Ohio, USA (Research topic: tRNA modification, editing and splicing in Trypanosoma brucei, Supervisor: Dr. Juan D. Alfonzo)
Mission of the lab: Our group studies various aspects of RNA biology of protozoan parasite Trypanosoma brucei and related flagellates. In those early evolved unicellular organisms most genes are post-transcriptionally regulated. Consequently, posttranscriptional processing of RNA becomes of a great importance in order to regulate complex life cycles of these pathogens. We are mainly interested in processes such as tRNA modifications, nuclear tRNA export and role of the only intron containing tRNA in trypanosomes. Our long term goal is an identification of unique mechanisms of RNA metabolism. We believe this will help us to reveal new drug targets to combat diseases caused by trypanosomatid parasites.
Queuosine: The role of an essential tRNA modification in parasitic protist Trypanosoma brucei
Transfer RNAs are typical for the large number of posttranscriptional modifications. Most of the tRNA modifications are present in the anticodon loop, which have crucial role in proper translation of proteins. Queuosine is one of the most complex tRNA modifications. Despite its omnipresence among bacteria and eukaryotes, role of queuosine tRNA modification is not clear. The main aim of this project is to evaluate the function and subunit composition of the enzyme responsible for queuosine formation in T. brucei. Using the RNAi knock-down strategy we want to address the principal question regarding the role of queuosine tRNA modification with respect to biology and physiology of this protozoan parasite.
Role of the only tRNA intron in trypanosomatids
In yeast Saccharomyces cerevisiae and other model organisms, 20% of all tRNAs contain introns. Their removal is an essential step in the maturation of tRNA precursors. In T. brucei, there is only one intron containing tRNA: tRNATyrGUA. Since this tRNA is responsible for decoding all tyrosine codons, intron removal is essential for viability. Using molecular and biochemical approaches, several non-canonical editing events were identified within the intron-containing tRNATyrGUA. The RNA editing involves guanosine-to-adenosine transitions (G to A) and an adenosine-to-uridine transversion (A to U), which are both necessary for proper processing of the intron. We have been obtaining tRNA intron sequences from our collection of newly identified trypanosomatid species. We hope this will help us to understand the process of RNA editing and ultimately identify biological function for the presence of the only intron containing tRNA in these organisms.
Nuclear export of tRNAs in trypanosomes
Regulation of tRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about tRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved; only a few orthologs can be easily identified in the genome of T. brucei. Thus, we are going to employ methods of molecular biology and biochemistry to identify and characterize the tRNA export machinery in trypanosomes.
Euglena gracilis as a new model organism
Euglena gracilisis the evolutionarily closest free-living photosynthetic relative of the kinetoplastid parasites that cause serious diseases including African sleeping sickness, leishmaniasis and Chagas disease. Hence, this protist provides an evolutionary link to understand the emergence of several unique molecular processes in kinetoplastids. Moreover, Euglena spp. is also an ecologically significant, yet severely understudied protist. Despite the efforts of several laboratories, the Euglena genome project has not yet been assembled and the lack of tools for forward and reverse genetics makes Euglena refractory for molecular studies. It was shown recently that electroporation of dsRNA into its cells leads to specific degradation of the target sequence, although this process is only temporary, being lost after few generations. In our lab, we are going to analyze the genome project data with the emphasis on mitochondrial proteome, RNA editing and processing, and make a comparative analysis with the genomes of the parasitic groups within the phylum Euglenozoa. Moreover we will design and establish a transfection protocol of E. gracilis with the aim to achieve over-expression from a stably integrated construct. Provided this phase was successful, we will also generate cell line with T7-TR background, representing a tool for functional analysis of genes of interest using the RNAi approach.