RNA importation into the mitochrondria of the kinetoplastid protozoan, Leishmania tarentolae

Trypanosomatid protozoa are parasites responsible for important human and animal diseases in tropical areas of the world. In the evolutionary tree of life, the trypanosomatids belong to one of the earliest diverging branches of mitochondria-containing eukaryotes. These parasites possess an assortment of unusual biological and biochemical properties. Among the many fascinating features is the ability of L. tarentolae mitochondria to import nucleus-encoded transfer RNAs. This phenomenon is the main focus of the research presented in this dissertation. I have investigated several aspects of the importation of RNAs into the mitochondrion. An in vitro RNA importation assay using isolated mitochondria from L. tarentolae was established. Although mitochondrial membrane proteins are responsible for the transport of the RNA into the mitochondrion, the in vitro import of RNA does not appear to require a membrane potential or any additional cytoplasmic factors. A variety of RNA substrates differing in sequence lengths and structures were investigated in terms of in vitro import. I also present evidence for C to U nucleotide modification editing at the first position of the anticodon of the nucleus-encoded mitochondrial imported tRNATrp(CCA). This editing event leads to the formation of a tRNATrp(UCA) which can decode the mitochondrial UGA codons as tryptophan. A separate study indicated that the in vivo substrate for tRNA import into the mitochondrion is the processed mature form of the tRNA. In addition, a specific nucleotide modification present in mitochondrial tRNA was shown to potentially act as a negative determinant of tRNA import. Finally, an in vitro selection method was used to isolate novel molecules which expand the upper size limits of RNAs imported into mitochondria. These mitochondrial aptamer RNAs (MARs) possess the ability to be imported into mitochondria both in vitro and in vivo.