The gene-dense genome of the eukaryotic amoeba D. discoideum features nearly 10% mobile DNA sequences, mainly retrotransposons. The two main retrotransposons, DIRS-1 and Skipper-1, belong to the family of Long-Terminal-Repeat (LTR) Elements.

We are investigating which functional components of the RNA interference (RNAi)-Machinery are involved in the regulation of these LTR-elements. DIRS-1 contains three open reading frames and to study the function of the encoded proteins we are expressing them in bacteria. Upon purification, the three proteins will be tested for their in vitro activities (nucleic acid binding, formation of multimers, enzymatic activities). The DIRS-1 GAG protein is expected to serve as structural component of virus-like particles (VLPs). We are aiming to visualize such DIRS-1 VLPs by using Fluorescence microscopy on GFP-fusion proteins of the GAG protein. We further are in the process of isolating VLPs aiming to analyse their content.

Upon exposure to external stimuli like bacteria, retrotransposons appear over-expressed in the amoeba. We are investigating how different bacteria or their surface structures modulate the expression of individual retrotransposons, and whether this is altered in certain mutant strains.

Figure: The mbsrI selection marker is positioned within DIRS-1 and disrupted by an intron. To monitor the uptake of the DIRS-1^bsr bearing plasmid it is co-transformed with a G418 resistant plasmid (pISAR) into D. discoideum strains and selected with G418. Upon integration of the DIRS-1^bsr plasmid, transformed cells harbour a DIRS-1^bsr sequence with an intron, referred to as ‘master element’. After a complete retrotransposition cycle the DIRS-1bsr is integrated into the genome (referred as ‘copy element’) and allows for the expression of a functional bsr mRNA, thus adding resistance to blasticidin to the cells. Presence of the copy element which represents successful retrotransposition is assessed by subsequent PCR.

The maturation of ribosomal RNA (rRNA) is complex and requires numerous processing events. We have analysed in D. discoideum the primary transcripts and identified at least ten precursor molecules from which the mature rRNAs are generated. With their sequences at hand, we subsequently determined the global 2’-O-methylation pattern. We plan to extend these studies by investigating the pseudouridylation (Ψ) patterns in the rRNA, both in axenic growth of the amoeba as well as during development.

Even more complex is the maturation of transfer RNA (tRNA) molecules, for which more than 100 different chemical modifications have been reported. Our interest focuses on modifications of tRNAs carrying glutamines (Q). Modifications in these tRNAs are essential for protein integrity: If certain modifications are absent, proteins have a high propensity for misfolding. In most organisms, longer Q stretches lead to misfolded and aggregated proteins even when the responsible tRNAs are fully modified. This, however, does not seem to be the case for D. discoideum, where polyQ stretches as long as 100 appear to be tolerated. We are aiming to understand the molecular basis for this unusual phenomenon.

RNA molecules play important roles on all levels of gene expression. Next to these functions, certain RNA molecules have the capacity to catalyse chemical reactions. Such RNA enzymes (Ribozymes) are widespread in nature and occur as several distinct classes. One of the best studied examples, the hammerhead ribozyme, was originally found in sub-viral plant pathogens. We identified members of this ribozyme family first in the genome of Arabidopsis thaliana, and later in many further genomes. Despite their wide occurrence, little is known about the biological function of such ribozymes. We aim to address this open question by applying a broad spectrum of molecular biology techniques.