Trypanosomes are parasitic protozoa responsible for health problems in developing countries, including those with a substantial U.S. presence. Along with being an important pathogen, Trypanosoma brucei offers an expanded molecular biology landscape that extends beyond that of the standard set of model organisms. Indeed, many unique biological processes have been discovered in this parasite. In particular, its giant mitochondrion encloses an unusual DNA structure, referred to as the kinetoplast, and unconventional RNA processing pathways. The kinetoplast carries a protein-coding maxicircle genome catenated to minicircles which encode a vast array of guide RNAs (gRNAs). A multitude of nuclear-encoded factors mediate the interactions between maxi- and minicircle transcripts to create functional mRNAs via nucleolytic processing, U- insertion/deletion RNA editing, and 32 polyadenylation. Editing reactions are catalyzed by the RNA editing core complex, RECC (20S editosome), while each step of the enzymatic cascade is directed by gRNAs. Although studies of RECC have shown impressive progress, until recently little was known about gRNA biogenesis, stabilization, recruitment to the editing process, and post-editing metabolic fate. We discovered that RNA editing TUTase 1 (RET1) is involved in the nucleolytic processing of gRNA precursors and that mature gRNAs are stabilized via binding to gRNA binding complex subunits 1 and 2 (GRBC1/2). In preliminary studies we identified GRBC1/2-associated proteins, including NUDIX hydrolases, DExD/H RNA helicases, RNA binding proteins, and polypeptides lacking identifiable domains. The overall complexity of GRBC likely exceeds that of the RECC, which consists of ~20 proteins. This proposal focuses on GRBC protein composition, mechanisms of gRNA binding and commitment to the editing process, and post-editing gRNA displacement. We hypothesize that interacting RECC and GRBC form an RNA editing holoenzyme, termed the 40S editosome, and propose to: 1) define core protein components and those involved in RNA-mediated contacts within GRBC; 2) determine the mechanisms of gRNA binding to GRBC1/2 as well as GRBC-RECC interaction; and 3) investigate the molecular basis of post-editing gRNA displacement.
Trypanosomatids are the causative agents of parasitic diseases in developing countries, including areas of substantial American presence. Available treatments are often toxic and ineffective, and the need for new drugs is pressing. Because the mitochondrial gene expression is vital for the parasite, targeting essential parasite-specific ribonucleoprotein complexes, such as the guide RNA binding complex (GRBC), is a potential approach toward a new generation of trypanocides.
