Trypanosoma brucei species inflict health hazards and economic hardship on arguably the most marginalized populations in the world. Some of the best-studied Excavata, trypanosomes also represent important models in many areas of research, including antigenic variation, host-pathogen interaction, developmental reprogramming and mitochondrial biology. This project will elucidate mechanisms by which macromolecular RNA editing substrate binding complex (RESC) stabilizes and delivers mitochondrial pre-mRNAs and guide RNAs into the U-insertion/deletion editing pathway, and coordinates polyadenylation and translation of edited mRNAs. We establish the RESC platform as the RNA binding constituent of the editing holoenzyme and seek to investigate its role in editing reactions, and functions beyond the RNA editing process. To this end, we demonstrate that RESC-associated MERS1 pyrophosphohydrolase and KPAP1 poly(A) polymerase target pre-mRNA 5? and 3? ends, respectively. Importantly, both 5? pyrophosphate removal and 3? A-tailing appear to be critical for pre-mRNA stabilization prior to editing. Conversely, specific module within RESC is suggested to couple the completion of editing with post-editing 3? A/U-tailing and mRNA binding to the ribosome. Collectively, the existing evidence positions the ~25 polypeptide RESC complex as the multimodal nexus of mitochondrial RNA processing. Furthermore, initial investigation of RESC-associated MERS1 complex, RNA polymerase (MTRNAP), and the 3? processome (MPsome) challenges the long-standing model of multicistronic maxicircle transcription and endonucleolytic partitioning of primary transcripts. The proposed experiments will deepen understanding of RNA editing by determining the RESC structure at near-atomic resolution and RNA binding specificities of individual subunits. We will test a broad functional hypothesis that discrete RESC modules coordinate completion of mRNA editing with 3? modification and translational activation. Finally, we put forward a fundamentally novel concept of monocistronic pre-mRNAs that are transcribed from individual promoters and shaped by 5? modification and antisense RNA-controlled 3?-5? degradation. By elucidating the RESC structure, RNA binding properties, and higher-order interactions, and evaluating the paradigm-shifting ?monocistronic hypothesis,? this program will expand the knowledge of critical parasite-specific processes and may provide new drug targets.
The goal of this project is to understand the mechanisms by which RNA editing substrate binding complex (RESC) orchestrates processing of mitochondrial transcripts in Trypanosoma brucei, a parasite responsible for devastating tropical diseases. Because these processes are unique to Trypanosomatids and required for the parasite's survival, the proposed studies will significantly expand the fundamental knowledge of essential molecular mechanisms in a medically and economically significant pathogen.
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