Unraveling the role of trypanosomal ncRNA in the regulation of antigenic variation

• Applicant: Professor Dr. Tim Nicolai Siegel
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277883612

The common need to evade the host immune response has led to the evolution of remarkably similar survival strategies even among evolutionarily distant organisms. One of these strategies is antigenic variation - the ability of an organism to periodically change the identity of the proteins displayed to the host immune system. Among the many infectious microorganisms that utilize mechanisms of antigenic variation to evade the host immune response some of the largest families of mutually exclusively expressed antigens are found in protozoan parasites such as Plasmodium falciparum, Giardia lamblia and Trypanosoma brucei, the causative agent of human sleeping sickness. The T. brucei genome codes for ~2500 isoforms of variant surface glycoproteins (VSGs). Roughly 10 million identical copies of VSGs form a dense surface coat that shields invariant parasite proteins from recognition by the host immune response. The mutually exclusive expression of different VSG isoforms and the periodic switch in expression from one VSG isoform to another permit the parasite to change its surface coat composition and, as a consequence, to evade the host immune response. While much has been learned about antigenic variation in T. brucei, the molecular mechanism leading to the transcriptional repression of one VSG and activation of another VSG isoform has remained elusive. Recent findings in P. falciparum and G. lamblia suggest that the tight regulation of antigenic variation is affected by the presence or absence of specific non-coding RNAs (ncRNA). Therefore, we have decided to investigate the role of ncRNA in antigenic variation in T. brucei. Following the establishment of RNA-sequencing and ribosome-profiling technology in T. brucei, which allowed us to perform genome-wide transcriptome and translatome analyses, we searched the genome for transcripts not translated into protein, i.e. putative ncRNA. Our data revealed that the region upstream of the actively transcribed vsg is transcribed into long ncRNA. Intriguingly, more than 15 years ago it was found that the same region that we find to be transcribed into long ncRNA contains a 'stabilizing element' crucial for continuous VSG expression and that short deletions in this region lead to a strong increase in VSG switching frequency. Based on our findings and the importance of ncRNA in ensuring mutually exclusive expression in other protozoan parasites, we hypothesize that the newly identified long ncRNA is required for antigenic variation in T. brucei. The goal of this work is to test this hypothesis and to elucidate the role of the region upstream of the vsg in stabilizing VSG expression. Taking advantage of the recently established CRISPR/Cas9 technology, we are for the first time able to perform marker-free genome-editing in T. brucei putting us in the unique position to manipulate regulatory elements like the region upstream of the vsg gene - a feature crucial for the success of the work proposed here.

• DFG Programme: Research Grants