ABSTRACT Flagellated kinetoplastid protozoans inflict public health hazards and economic burden predominately on marginalized populations in the developing world. The agents of African human and animal trypanosomiasis, Trypanosoma brucei sp., cause some of the most-studied, but least manageable and curable parasitic infections. Failing vector control, lack of vaccines and toxic therapeutics place the onus on identification, molecular understanding and validation of new drug targets. From the fundamental science perspective, these early branching Excavata represent valuable models of mitochondrial RNA biology, antigenic variation, host-pathogen interaction, post-transcriptional regulation, and other processes. Major findings in the previous funding period position a family of RNA binding pentatricopeptide repeat (PPR, 35 amino acids) proteins as the central conduit controlling mitochondrial gene expression. The unique ability of helix-turn-helix repeats to assemble into superhelical arrays recognizing extended and compositionally diverse RNA sequences enables the essential roles of more than 40 PPRs. Often including trypanosome-specific domains, these RNA “sequence readers” commit otherwise non-specific modification enzymes to their substrates, or block RNA degradation and extension. This project will elucidate the mechanisms by which nuclear-encoded PPRs govern mitochondrial mRNA biogenesis and translation via recruiting enzymatic complexes and ribosomes to transcripts at defined processing stages. We establish three priority areas focusing on PPRs that enable mRNA 5′ end modification, 3′ adenylation/uridylation, and translation. Building on our recent discoveries of gene-specific transcription initiation and exonucleolytic pre-mRNA processing, we identified PPR factors acting as “protein cap,” “editing sensor,” poly(A) binding protein, and potential translational activators. Our goal is to dissect the molecular machinery integrating these functions and to decipher principles of RNA recognition by repeat-containing proteins. By elucidating their RNA binding sites, structures, interactions, and outcomes of genetic alteration and protein engineering, this program builds on our momentum in a significant field and expands the knowledge of critical pathogen-specific processes.

PROJECT NARRATIVE The goal is to understand the mechanisms by which nuclear-encoded RNA binding pentatricopeptide repeat (PPR, 35 amino acids) proteins orchestrate mitochondrial mRNA biogenesis and translation in Trypanosoma brucei. Because PPR-mediated elements of these processes are unique to a parasite responsible for devastating diseases, the proposed studies will expand the fundamental knowledge of essential molecular mechanisms in a medically and economically significant pathogen.