: Trypanosomatids are causative agents of major diseases affecting developing countries around the world. Uridine insertion/deletion editing of mitochondria! mRNAs is unique to these parasites and is necessary for their viability. Our long-term goal is to elucidate the mechanisms of two key editing reactions: posttranscriptional formation of the 3' oligo[U] tail in guide RNAs and U-insertions into mRNA. These reactions are catalyzed by distinct RET1 and RET2 terminal uridylyltransferases (TUTases). We hypothesize that these enzymes share the mechanism of UTP recognition and transfer but their adaptation for a particular function is conferred by additional non-catalytic domains or interacting proteins within editing complexes. Furthermore, multiple uridylyltransferases may exist in Kinetoplastids in addition to those involved in editing. Thus, an inhibitor of trypanosomal UTP-specific RNA transferases may potentially become a multi-target therapeutic agent.
The specific aims of this proposal are: 1. Define the mechanisms that regulate RET1 activity during the 3' end processing of guide RNAs. The average length of the oligo[U] tail in vivo is -15 nucleotides whereas in vitro recombinant RET1 adds hundreds of uridines to the synthetic guide RNA. We will isolate factors that modulate RET1 activity in the mitochondria and assess interaction of guide RNA 3' end processing and RNA editing complexes. Ultimately, reconstruction of the regulated uridylylation of short RNAs in vitro will be performed. 2. Characterize the mechanism of U-insertion editing. Uridines are inserted into the mRNA by RET2, an integral part of the enzymatic cascade within the core editing complex. A set of RNA-protein and nucleotideprotein cross-linking agents will be utilized to investigate the recognition of UTP and RNA substrates by RET2. Catalytic and RNA binding domain swapping between RET1 and RET2 will be used to understand the structural basis of specificity and processivity of U-addition. 3. Identify novel RNA uridylyltransferases. To expand our knowledge of TUTase-specific features, genome databases will be searched with the RET1 and RET2 sequence profiles. Highest-scoring homologues will be expressed and uridylyltransferase activity assayed. Sequences of the newly identified UTP-specific transferases will be used to establish characteristic protein motifs for this family of enzymes.
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